Small interfering RNAS with target-specific seed sequences

ABSTRACT

Disclosed are methods for design and synthesis of siRNA libraries, siRNA libraries produced thereby, siRNA molecules, and uses thereof.

RELATED APPLICATION INFORMATION

This application claims priority under 35 U.S.C. §371 from PCTApplication No. PCT/US2012/025004, filed Feb. 14, 2012, which claims thebenefit of U.S. Provisional Application No. 61/442,765, filed Feb. 14,2011, the disclosures of which are incorporated by reference herein intheir entirety.

FIELD OF THE INVENTION

The invention relates to methods for designing small interfering RNAs(siRNAs) based upon enrichment of target-specific siRNA sequences,siRNAs produced thereby, and methods for using the same. Moreparticularly, the invention relates to small interfering RNAs havingactivity against pests or pathogens and their use in plants.

BACKGROUND

In the past decade, RNA interference (RNAi) has been described andcharacterized in organisms as diverse as plants, fungi, nematodes,hydra, and humans. Zamore and Haley (2005) Science 309, 1519-24. RNAinterference in plants is commonly referred to as post-transcriptionalgene silencing or RNA silencing and is referred to as quelling in fungi.The process of post-transcriptional gene silencing is thought to be anevolutionarily conserved cellular defense mechanism used to prevent theexpression of foreign genes and is commonly shared by diverse flora andphyla. Fire (1999) Trends Genet. 15, 358-363.

RNA interference occurs when an organism recognizes double-stranded RNAmolecules and hydrolyzes them. The resulting hydrolysis products aresmall RNA fragments of 19-24 nucleotides in length, called smallinterfering RNAs (siRNAs) or microRNAs (miRNAs). The siRNAs then diffuseor are carried throughout the organism, including across cellularmembranes, where they hybridize to mRNAs (or other RNAs) and causehydrolysis of the RNA. Interfering RNAs are recognized by the RNAinterference silencing complex (RISC) into which an effector strand (or“guide strand”) of the RNA is loaded. This guide strand acts as atemplate for the recognition and destruction of the duplex sequences.This process is repeated each time the siRNA hybridizes to itscomplementary-RNA target, effectively preventing those mRNAs from beingtranslated, and thus “silencing” the expression of specific genes. Mostplant miRNAs show extensive base pairing to, and guide cleavage of theirtarget mRNAs. Jones-Rhoades et al. (2006) Annu. Rev. Plant Biol. 57,19-53; Llave et al. (2002) Proc. Natl. Acad. Sci. USA 97, 13401-13406.In other instances, interfering RNAs may bind to target RNA moleculeshaving imperfect complementarity, causing translational repressionwithout mRNA degradation. The majority of the animal miRNAs studied sofar appear to function in this manner.

Based upon the role of miRNAs as endogenous regulators of geneexpression, substantial efforts have been made toward the design ofmiRNAs for targeted regulation of gene expression. For example,pre-miRNAs can be designed by replacing both the 21-nucleotide maturemiRNA sequence and the complementary sequence (i.e., the miRNA* strandor miRNA star strand), with engineered or synthetic 21-nucleotidesequences. Such artificial pre-miRNAs have sequences identical to thoseof the natural pre-miRNAs except in the region encoding the mature miRNAand the star strand. By this method, artificial miRNAs (amiRNA) havebeen designed that can target and silence specific mRNA transcripts withcomplementary sequences.

Within miRNA sequences, highly conserved regions of 6-7 nucleotides,which are called seed sequences, are responsible for base pairing with atarget gene/RNA. The seed sequences are positioned at nucleotides 2-7 or2-8 by linear counting from the 5′-end of the miRNA molecule, while theremaining nucleotides are called non-seed sequences. miRNAs that aremembers of a same miRNA family (i.e., miRNAs with the same sequence atnucleotides 2-8) share the same predicted mRNA targets. See Bartel(2009) Cell 136, 215-233.

Given their role in sequence-specific gene regulation, siRNAs areenvisioned to have many applications, including studies of genefunction, development of therapies for conditions associated withaberrant protein expression or accumulation, and methods for conferringdesirable traits, including in plants. To meet this need, the inventionprovides methods for efficient design of target-specific siRNAs, siRNAlibraries, and siRNA molecules produced thereby, and methods for usingthe same.

SUMMARY

The invention described herein is a method of preparing a library ofsmall interfering RNAs (siRNAs), siRNAs produced thereby, and usesthereof.

One aspect of the invention is a method of preparing a library of smallinterfering RNAs (siRNAs) comprising synthesizing a plurality of RNAmolecules, wherein each RNA molecule comprises (a) a seed sequencecomprising random nucleotides, and (b) a non-seed sequence comprisingdesignated nucleotides.

Another aspect of the invention is a method of preparing a library ofsmall interfering RNAs (siRNAs) comprising synthesizing a plurality ofRNA molecules, wherein each RNA molecule comprises (a) a seed sequencecomprising nucleotides representative of one or more microRNA seedsequences of a target organism or an organism related to the targetorganism, and (b) a non-seed sequence comprising designated nucleotides.

Another aspect of the invention is a method for preparing a library ofsmall interfering RNAs (siRNAs), further comprising the steps of: (a)excluding RNA molecules comprising one or more nucleotides within theseed sequence, which occur at low frequency at corresponding positionsof microRNA sequences of a target organism or an organism related to atarget organism; (b) excluding RNA molecules comprising a homonucleotidequadruplet within the seed sequence; and/or (c) excluding RNA moleculescomprising a seed sequence having greater GC content at positions 1-9than the GC content at positions 11-19.

Another aspect of the invention is a siRNA library comprising aplurality of RNA molecules, wherein each RNA molecule comprises (a) aseed sequence comprising random nucleotides, and (b) a non-seed sequencecomprising designated nucleotides. Another aspect of the invention is asiRNA library comprising a plurality of RNA molecules, wherein each RNAmolecule comprises (a) a seed sequence comprising nucleotidesrepresentative of one or more microRNA seed sequences of a targetorganism or an organism related to the target organism, and (b) anon-seed sequence. In one aspect, the siRNA library is an in silicosiRNA library.

Another aspect of the invention comprises a siRNA library wherein thenon-seed sequence comprises a consensus microRNA sequence from a plantpest or pathogen or from an organism related to a plant pest orpathogen. One aspect of the invention is a siRNA library, wherein thenon-seed sequence comprises nucleotides occupying positions 1, and 9-19of SEQ ID NO: 47. Another aspect of the invention is a siRNA library,wherein the non-seed sequence comprises nucleotides occupying positions1, and 9-19 of SEQ ID NO: 50. Another aspect of the invention is a siRNAlibrary, wherein the non-seed sequence further comprises one or morenucleotide substitutions to improve microRNA stability.

Another aspect of the invention is a siRNA library, which excludes: (a)RNA molecules comprising one or more nucleotides within the seedsequence, which occur at low frequency at corresponding positions ofmicroRNA sequences of a target organism or an organism related to atarget organism; (b) RNA molecules comprising a homonucleotidequadruplet within the seed sequence; and/or (c) RNA molecules comprisinga seed sequence having greater GC content at positions 1-9 than atpositions 11-19.

Another aspect of the invention is a siRNA library, which excludes RNAmolecules comprising a seed sequence complementary to a host nucleicacid. An additional aspect of the invention is a siRNA library, whichcomprises a seed sequence of residues 2-8 of SEQ ID NO: 49. Anotheraspect of the invention is a siRNA library, which comprises a seedsequence of residues 2-8 of SEQ ID NO: 51.

Another aspect of the invention is a siRNA molecule comprising (a) aseed sequence comprising nucleotides representative of one or moremicroRNA seed sequences of a target organism or an organism related tothe target organism, and (b) a non-seed sequence. Another aspect of theinvention is a siRNA molecule which comprises at least about 19nucleotides, and wherein the seed sequence comprises 6-8 nucleotides.One aspect of the invention is a siRNA molecule, which comprises 21nucleotides, wherein the seed sequence comprises nucleotides occupyingpositions 2-8 of the RNA molecule, and wherein the non-seed sequencecomprises nucleotides occupying positions 1 and 9-21 of the RNAmolecule.

Another aspect of the invention is a siRNA molecule, wherein thenon-seed sequence comprises a consensus microRNA sequence.

Another aspect of the invention is a siRNA molecule, wherein thenon-seed sequence comprises a consensus microRNA sequence from a plantpest or pathogen, or from an organism related to a plant pest orpathogen. One aspect of the invention is a siRNA molecule, wherein thenon-seed sequence comprises nucleotides occupying positions 1, and 9-19of SEQ ID NO: 49.

Another aspect of the invention is a siRNA molecule, wherein thenon-seed sequence comprises nucleotides occupying positions 1, and 9-19of SEQ ID NO: 51. Another aspect of the invention is a siRNA molecule,wherein the non-seed sequence further comprises one or more nucleotidesubstitutions to improve microRNA stability.

Another aspect of the invention is a siRNA molecule, which comprises aseed sequence of residues 2-8 of SEQ ID NO: 49. Another aspect of theinvention is a siRNA molecule, which comprises a seed sequence ofresidues 2-8 of SEQ ID NO: 48. A further aspect of the invention is asiRNA molecule which comprises a seed sequence of residues 2-8 of anyone of SEQ ID NOs: 1-14. An additional aspect of the invention is asiRNA molecule, which comprises a seed sequence of residues 2-8 of SEQID NO: 51.

Another aspect of the invention is a siRNA molecule, which comprises thenucleotide sequence of any one of SEQ ID NOs: 1-14. Another aspect ofthe invention is a siRNA molecule, which comprises the nucleotidesequence of SEQ ID NO: 51.

Another aspect of the invention is an artificial RNA molecule comprisingthe siRNA molecule, which comprises the nucleotide sequence of any oneof SEQ ID NOs: 16-29. A further aspect of the invention is an artificialRNA molecule comprising the siRNA molecule, which comprises thenucleotide sequence of SEQ ID NO: 51.

Another aspect of the invention is a vector comprising the siRNAmolecule, which comprises the nucleotide sequence of any one of SEQ IDNOs: 31-44. An additional aspect of the invention is a vector comprisingthe siRNA molecule, which comprises the nucleotide sequence of SEQ IDNO: 51

Another aspect of the invention is a transgenic plant, or part thereof,comprising the siRNA molecule of any one of SEQ ID NOs: 1-51. In oneaspect, the transgenic plant is Glycine max. In another aspect, thetransgenic plant is Zea mays.

Another aspect of the invention is a plant product comprising the siRNAmolecule of any one of SEQ ID NOs: 1-51. A further aspect of theinvention is a commodity product comprising the siRNA of any one of SEQID NOs: 1-51.

Another aspect of the invention is a method of identifying a siRNA thatconfers a desirable phenotypic outcome in a target organism comprising:(a) contacting the target organism with a siRNA molecule of a siRNAlibrary; and (b) correlating the siRNA treatment of (a) with thedesirable phenotypic outcome. Another aspect of the invention is amethod of identifying a siRNA that confers resistance to soybean cystnematode comprising: (a) contacting soybean cyst nematode with a siRNAmolecule of a siRNA library; and (b) correlating the siRNA treatment of(a) with soybean resistance to soybean cyst nematode infection. Afurther aspect of the invention is a method of identifying a siRNA thatconfers resistance to corn rootworm: (a) contacting corn rootworm with asiRNA molecule of a siRNA library; and (b) correlating the siRNAtreatment of (a) with corn resistance to corn rootworm infection.

Another aspect of the invention is a method of conferring nematoderesistance to a plant comprising expressing in the plant a nucleic acidcomprising a siRNA of SEQ ID NOs: 1-14, whereby the plant is nematoderesistant. Another aspect of the invention is a method of conferringinsect resistance to a plant comprising expressing in the plant anucleic acid comprising a siRNA of SEQ ID NO: 51, whereby the plant isinsect resistant.

Another aspect of the invention is a method of reducing nematodeinfectivity to a plant comprising contacting the nematode with a nucleicacid comprising a siRNA of SEQ ID NOs: 1-14, whereby nematodeinfectivity is reduced. Another aspect of the invention is a method ofreducing insect infectivity to a plant comprising contacting the insectwith a nucleic acid comprising a siRNA of SEQ ID NO: 51, whereby insectinfectivity is reduced.

An aspect of the invention is a method of reducing risk of nematodeinfection in a plant comprising expressing in the plant a nucleic acidcomprising a siRNA of SEQ ID NOs: 1-14, whereby risk of nematodeinfection is reduced. Another aspect of the invention is a method ofreducing risk of nematode infection in a plant comprising expressing inthe plant a nucleic acid comprising a siRNA of SEQ ID NO: 51, wherebyrisk of insect infection is reduced.

Another aspect of the invention is a method of providing a grower with ameans controlling nematode pests comprising supplying seed to a grower,wherein the seed comprises a nucleic acid comprising a siRNA of SEQ IDNOs: 1-14. A further aspect of the invention is a method of providing agrower with a means controlling insect pests comprising supplying seedto a grower, wherein the seed comprises a nucleic acid comprising asiRNA of SEQ ID NO: 51.

Another aspect of the invention is a siRNA molecule that targets both anematode gene and an endogenous plant gene related to anematode-resistant plant phenotype.

Another aspect of the invention is a transgenic plant, or part thereof,having a reduced level of expression of a ethylene response genecompared to a non-transgenic plant of the same species, wherein thetransgenic plant comprises an siRNA that suppresses the expression of apest nematode gene, and wherein the transgenic plant has a greatertolerance to infection by the nematode pest than would be expected fromthe reduced level of expression of the ethylene response gene or thesuppression of the nematode gene alone.

Another aspect of the invention is a method of enhancing resistance of aplant, or part thereof, to infection by a nematode pest, comprisingintroducing into the plant, or part thereof, a nucleic acid comprising asiRNA molecule that suppresses the expression of a nematode gene therebyreducing the ability of the nematode to infect the plant, or partthereof, wherein the plant, or part thereof, additionally has a reducedlevel of expression of an ethylene response gene compared to a plant, orpart thereof, of the same species without the siRNA molecule, wherebythe plant, or part thereof, comprising the siRNA has a greaterresistance to infection by the nematode than would be expected from thesuppression of the nematode gene or the suppression of the ethyleneresponse gene alone.

BRIEF DESCRIPTION OF THE SEQUENCES

The Sequence Listing provides disclosure of siRNAs and amiRNAs of thefollowing sequences that are specific aspects of the invention.

SEQ ID NOs: 1-15 are the nucleic acid sequences of siRNAs, which arealso listed in Table 8, below.

SEQ ID NOs: 16-30 are the nucleic acid sequences of amiRNAs, which arealso listed in Table 9, below.

SEQ ID NOs: 31-45 are the nucleic acid sequences of amiRNAs inexpression vectors, which are also listed in Table 10, below. SEQ ID NO:46 is the empty expression vector control.

SEQ ID NO: 47, 5′-UNNNNNNNUGUUGAUCUGGUU-3′, is the sequence of a siRNAcontaining a random seed sequence and a non-seed sequence that is aconsensus of C. elegans miRNAs, as described in Example 1, below.

SEQ ID NO: 48, 5′-URDSDKVDUGUUGAUCUGGUU-3′, encompasses the sequences ofall siRNAs in the enriched library, prepared as described in Example 1,below.

SEQ ID NO: 49, 5′-URDBDKVDUGUUGAUCUGGUU-3′, encompasses the sequencesfor siRNAs having activity against soybean cyst nematode, as describedin Example 4, below.

SEQ ID NO: 50, 5′-UNNNNNNNUAUCCGGAUUCUU-3′, is the sequence of a siRNAcontaining a random seed sequence and a non-seed sequence that is aconsensus of Tribolium castaneum miRNAs, as described in Example 6,below.

SEQ ID NO: 51, 5′-UNDNWDNNUAUCCGGAUUCUU-3′, encompasses the sequences ofall siRNAs in the enriched library, prepared as described in Example 6,below.

SEQ ID NO: 52 is the nucleotide sequence of a soybean ETR1 nucleic acid(gma-ETR1), GenBank accession number EF210138.

SEQ ID NO: 53 is the nucleotide sequence comprising the mRNA portion ofa soybean ETR1 that binds to siRNA0097 and siRNA0145.

SEQ ID NO: 54 is the nucleotide sequence comprising the mRNA portion ofa soybean ETR1 that binds to siRNA0097* and siRNA0145*.

SEQ ID NO: 55 is the nucleotide sequence of siRNA0097*.

SEQ ID NO: 56 is the nucleotide sequence of siRNA0145*.

SEQ ID NO: 57 is the nucleotide sequence describing the mRNA sequence ofa soybean ETR1 that has low complementarity to amiRNA0043*.

SEQ ID NO: 58 is the nucleotide sequence describing the mRNA sequence ofa soybean ETR1 that has low complementarity to amiRNA0046*.

SEQ ID NO: 59 is the nucleotide sequence of siRNA0043*.

SEQ ID NO: 60 is the nucleotide sequence of siRNA0046*.

DETAILED DESCRIPTION

The invention provides methods for preparing a library of smallinterfering RNAs (siRNAs), libraries produced by the methods, andindividual siRNA molecules. As described herein, the library designincludes enrichment of siRNAs having target-specific sequences. Thelibraries are useful for selecting one or more siRNAs that elicit adesired phenotype when contacted with a target organism. Also providedare siRNAs produced thereby and methods for using the same.

siRNA Molecules

The invention disclosed herein provides a strategy for the design ofsiRNAs having activity in a target organism. Also provided are siRNAsproduced thereby, which have utility for numerous applications, asdescribed herein below. The scope of the invention is not limited tonucleic acids or libraries comprising siRNAs for which specificsequences are disclosed herein. Rather, sequences from any organism,both known and presently unknown, can be used to design siRNAs accordingto the disclosed methods.

The term “RNA” includes any molecule comprising at least oneribonucleotide residue, including those possessing one or more naturalribonucleotides of the following bases: adenine, cytosine, guanine, anduracil; abbreviated A, C, G, and U, respectively, modifiedribonucleotides, and non-ribonucleotides. “Ribonucleotide” means anucleotide with a hydroxyl group at the 2′ position of theD-ribofuranose moiety.

As used herein, the terms and phrases “RNA,” “RNA molecule(s),” and “RNAsequence(s),” are used interchangeably to refer to RNA that mediates RNAinterference. These terms and phrases include single-stranded RNA,double-stranded RNA, isolated RNA, partially purified RNA, essentiallypure RNA, synthetic RNA, recombinant RNA, intracellular RNA, and RNAthat differs from naturally occurring RNA by the addition, deletion,substitution, and/or alteration of one or more nucleotides. “mRNA”refers to messenger RNA, which is RNA produced by transcription.

An “interfering RNA” (e.g., siRNA and miRNA) is a RNA molecule capableof post-transcriptional gene silencing or suppression, RNA silencing,and/or decreasing gene expression. Interfering RNAs affectsequence-specific, post-transcriptional gene silencing in animals andplants by base pairing to the mRNA sequence of a target nucleic acid.Thus, the siRNA is at least partially complementary to the silencedgene. The partially complementary siRNA may include one or moremismatches, bulges, internal loops, and/or non-Watson-Crick base pairs(i.e., G-U wobble base pairs).

The terms “silencing” and “suppression” are used interchangeably togenerally describe substantial and measurable reductions of the amountof mRNA available in the cell for binding and decoding by ribosomes. Thetranscribed RNA can be in the sense orientation to effect what isreferred to as co-suppression, in the anti-sense orientation to effectwhat is referred to as anti-sense suppression, or in both orientationsproducing a double-stranded RNA to effect what is referred to as RNAinterference. A “silenced” gene refers to a gene that is subject tosilencing or suppression of the mRNA encoded by the gene.

The descriptions “small interfering RNA” and “siRNA” are usedinterchangeably herein to describe a synthetic or non-naturalinterfering RNA. The terms “miRNA” or “microRNA” generally refer tonatural or endogenous interfering RNAs. As used herein, “miRNA” refersto interfering RNAs that have been or will be processed in vitro or invivo from a pre-microRNA precursor to form the active interfering RNA.Both siRNAs and miRNAs are RNA molecules of about 19-24 nucleotides,although shorter or longer siRNAs/miRNAs, e.g., between 18 and 26nucleotides in length, may also be useful. siRNAs or miRNAs may besingle stranded or double stranded.

microRNAs are encoded by genes that are transcribed but not translatedinto protein (non-coding DNA), although some miRNAs are encoded bysequences that overlap protein-coding genes. miRNAs are processed fromprimary transcripts known as pri-miRNAs to short stem-loop structurescalled pre-miRNAs that are further processed creating functionalsiRNAs/miRNAs. Typically, a portion of the precursor miRNA is cleaved toproduce the final miRNA molecule. The stem-loop structures may rangefrom, for example, about 50 to about 80 nucleotides, or about 60nucleotides to about 70 nucleotides (including the miRNA residues, thosepairing to the miRNA, and any intervening segments). The secondarystructure of the stem-loop structure is not fully base-paired;mismatches, bulges, internal loops, non-Watson-Crick base pairs (i.e.,G-U wobble base pairs), and other features are frequently observed inpre-miRNAs and such characteristics are thought to be important forprocessing. Mature miRNA molecules are partially complementary to one ormore messenger RNA molecules, and they function to regulate geneexpression. siRNAs of the invention have structural and functionalproperties of endogenous miRNAs (e.g., gene silencing and suppressivefunctions). Thus, in various aspects of the invention, siRNAs of theinvention can be processed from a portion of a precursor transcriptthat, optionally, folds into a stable hairpin (i.e., a duplex) or astem-loop structure.

The phrases “target-specific small interfering RNAs,” “target-specificsiRNAs,” “target-specific microRNAs,” “target-specific miRNAs,”“target-specific amiRNAs,” and “target-specific nucleotide sequence”refer to interfering RNAs that have been designed to selectivelyhybridize with nucleic acids in a target organism but not in anon-target organism, such as a host organism (the organism expressing orproducing the miRNA) or a consumer of the host organism. Consequently,“target-specific siRNAs or amiRNAs” only produce phenotypes in targetorganisms and do not produce phenotypes in non-target organisms.

In one aspect of the invention, a siRNA molecule comprises (a) a seedsequence comprising nucleotides representative of one or more microRNAseed sequences of a target organism or an organism related to the targetorganism, or a consensus sequence thereof, and (b) a non-seed sequence.Such siRNA molecules comprise at least about 19 nucleotides, wherein theseed sequence comprises 6-7 nucleotides.

The description “seed sequence,” as used herein, refers to a region of asiRNA molecule that is at least partially complementary to a target geneor RNA. As used herein, the seed sequence consists of 6-7 nucleotidesbeginning at the second residue from the 5′-end of a siRNA (e.g.,nucleotides 2-7 or 2-8, as numbered linearly from the 5′-end of asiRNA). The seed sequences are the most highly conserved regions amongmetazoan miRNAs, and miRNAs with the same sequence at nucleotides 2-8share the same predicted mRNA targets. See Bartel (2009) Cell 136,215-233.

In one aspect of the invention, nucleotides within the seed sequence arebased upon the frequency at which particular nucleotides are observed inmiRNA seed sequences of a “target organism,” i.e., an organism in whicha siRNA of the invention is intended to be functional for genesilencing. Similarly, nucleotides within the seed sequence may be basedupon the frequency at which particular nucleotides are observed in miRNAseed sequences of an “organism related to a target organism.” In thiscontext, “related” means relative phylogenic closeness between and amongorganisms, whether evolutionary relationships are determined byphenotypic traits, molecular markers, and/or variation in rates ofspeciation and/or extinction, or sequence identity or similarity. Thedegree of relation may be in some aspects, closely related throughphylogeny, such as sharing the same genus or family. In other aspects,the degree of phylogenic relation may be distant, such as sharing onlythe same phylum or class. In other aspects, there may be no phylogenicrelation to target an organism but the non-seed sequence may be“related” to the target organism through sequence homology, similarity,or identity. The consensus non-seed sequence can also be prepared fromnon-seed sequences from the target organism and/or from one or moreorganisms related to the target organism. As used herein, any organismthat contains nucleic acids capable of interacting with seed sequencesof the invention disclosed herein is a “target” organism.

The nucleotides of the seed sequence may be further selected based uponobserved frequencies at each position in naturally occurring miRNAs, forexample, by excluding those nucleotides that are observed at a lowfrequency. A low frequency can comprise an observed incidence of lessthan about 50% among a population of naturally occurring miRNAs, or lessthan about 45%, or less than about 40%, or less than about 35%, or lessthan about 30%, or less than about 25%, or less than about 20%, or lessthan about 15%, or less than about 10%, or less than about 5%.

The seed sequence may alternatively comprise a consensus of two or moremiRNA seed sequences of a target organism and/or an organism related tothe target organism. See also Example 1. The phrase “consensussequence,” as used herein, refers to a nucleotide sequence wherein eachnucleotide represents the most frequently observed nucleotide at aparticular position in the sequence when similar or related sequencesare compared to each other as described herein for determining thesimilarity or identity (see below). As used herein, the consensussequence of a siRNA, or part thereof, refers to either a selected groupof siRNAs or all siRNAs that are conserved within an organism, species,genus, family, order, class, phylum, kingdom, or domain. The term“consensus” also encompasses structural elements known or predicted fromthe sequence, or from analogous or homologous sequences, such asduplexes, mismatches, budges, G-U wobble base pairs, loops, hairpins,tetraloops, inter alia, which are observed in pri-mRNA, pre-miRNA,miRNA, or siRNA sequences that are thought to be important for miRNAprocessing. See, e.g., Saxena et al. (2003) J. Biol. Chem. 278,44312-44319.

Representative siRNA seed sequences of the invention include residues2-8 of SEQ ID NO: 48, which is a degenerate consensus sequence, thedegeneracy based upon the frequency of nucleotides observed at the samepositions in naturally occurring miRNAs of C. elegans. Additionalrepresentative seed sequences include residues 2-8 of any one of SEQ IDNOs: 1-14, and residues 2-8 of SEQ ID NO: 49, which is a consensus ofthe seed sequences of SEQ ID NOs: 1-14. In addition, SEQ ID NO: 51, is adegenerate consensus sequence, the degeneracy based upon the frequencyof nucleotides observed at the same positions in naturally occurringmiRNAs of Tribolium castaneum.

The description “non-seed sequence,” as used herein, refers to allsequences of a siRNA or miRNA that are not the seed sequence. For a21-nucleotide siRNA, the non-seed sequence comprises linear nucleotides1 and 8-21 or 1 and 9-21, depending on whether the seed sequenceconsists of 6 nucleotides (e.g., positions 2-7) or 7 nucleotides (e.g.,positions 2-8). In one aspect of the invention, the non-seed sequencecomprises a naturally occurring miRNA non-seed sequence. In anotheraspect of the invention, the non-seed sequence comprises a consensusmicroRNA non-seed sequence, i.e., a consensus of miRNA non-seedsequences. Such a consensus may be prepared from two or more miRNAnon-seed sequences, for example, three miRNA sequences, or four miRNAsequences, or five miRNA sequences, or six miRNA sequences, or sevenmiRNA sequences, or eight miRNA sequences, or nine miRNA sequences, orten miRNA sequences, or twenty miRNA sequences, or thirty miRNAsequences, or forty miRNA sequences, or fifty miRNA sequences, or more.One skilled in the art understands techniques and computational toolsfor making such alignments and can readily prepare consensus sequencesusing any number of miRNA sequences.

In one aspect of the invention, the miRNA non-seed sequence or consensusof miRNA non-seed sequences comprises a consensus of non-seed sequencesfrom a target organism, i.e., an organism in which a siRNA of theinvention is intended to be functional for gene silencing. Similarly,the consensus of miRNA non-seed sequences can comprise a consensus ofnon-seed sequences related to the target organism. In this context,“related” means relative phylogenic closeness between or amongorganisms, as described herein above with respect to design of seedsequences.

In another aspect of the invention, the non-seed sequence is partiallyor completely synthetic, i.e., a non-naturally occurring sequence thatshows desired functional properties as determined by modeling orempirically. For example, the non-seed sequence can comprise one or morenucleotide substitutions relative to a naturally occurring miRNAsequence, a siRNA sequence, or a miRNA/siRNA consensus sequence toimprove siRNA stability, such as 3′-terminal uridines or deoxythymidine.See Example 1.

For example, where the target organism is a plant parasitic nematode, auseful non-seed sequence can comprise a consensus of miRNA non-seedsequences of the model nematode Caenorhabditis elegans. A representativenon-seed sequence having these properties is set forth as nucleotides 1and 9-19 of SEQ ID NO: 47. See Example 1. As another example, where thetarget organism is a plant parasitic nematode, other useful seedsequences include consensus sequences of miRNA non-seed sequences of oneor more of the nematodes identified in Table 6. As a further example,where the target organism is an insect pest, a useful non-seed sequencecan comprise consensus of miRNA non-seed sequences of the organismTribolium castaneum. A representative non-seed sequence having theseproperties is set forth as nucleotides 1 and 9-19 of SEQ ID NO: 50. SeeExample 6.

Representative siRNAs of the invention include SEQ ID NOs: 1-14, whichwere obtained from the siRNA library described in Example 1. AdditionalsiRNA molecules of the invention include molecules of siRNA librariesproduced by the methods described herein below.

The invention also provides “artificial microRNAs” or “amiRNAs,” whichare non-naturally occurring nucleic acid sequences that are capable ofexpressing siRNA molecules. In one aspect of the invention, the sequenceof the Glycine max miRNA precursor gma-MIR164 was used as the startingsequence or backbone for designing an artificial microRNA targetingnematodes that will be expressed in a plant host. The design of thisartificial microRNA for use in soybeans is described in U.S. ProvisionalApplication 61/421,275 and a similar approach for use of amiRNAs inArabidopsis thaliana is described by Schwab et al. (2006) Plant Cell 18,1121-1133, both of which are incorporated herein by reference in theirentirety. Representative amiRNAs of the invention include amiRNAscomprising a siRNA of any one of SEQ ID NOs: 1-14, for example, theamiRNAs set forth as SEQ ID NOs: 16-29.

The above-described siRNAs, or seed or non-seed sequences therein, orprecursors thereof (e.g., pri-miRNA and pre-miRNA), may be furtheraltered by the addition, deletion, substitution, and/or alteration ofone or more nucleotides to introduce variation; to modify specificity;to alter complementarity; to introduce or remove secondary structuralelements such as mismatches, bulges, loops, single-stranded regions,double-stranded regions, overhangs, or other motifs; to enhance ormaintain the capability of the RNA to be processed in a RISC complex invitro or in vivo; to improve the stability of the RNA molecule in vitroor in vivo (i.e., the ability of the RNA molecule to be maintainedwithout being degraded by nucleases and/or its ability to fold intostable secondary or tertiary structures); and/or to enhance the abilityto hybridize to a target gene/RNA.

Nucleic acids that share a substantial degree of complementarity willform stable interactions with each other, for example, by matching basepairs. The terms “complementary” or “complementarity” refer to thespecific base pairing of nucleotide bases in nucleic acids. The phrase“perfect complementarity,” as used herein, refers to complete (100%)base paring within a contiguous region of nucleic acid, such as betweena seed sequence in a siRNA and its complementary sequence in a targetgene/RNA, as described herein. “Partial complementarity” or “partiallycomplementary” indicates that two sequences can base pair with oneanother, although the complementarity is not 100%. As used herein, thephrase “sequence complementary to a sequence” is used to describe anucleotide sequence capable of base pairing with another sequence,although the complementarity may not be 100%.

Alternatively stated, the phrase “sequence complementary to a sequence”with respect to two nucleotide sequences indicates that thetwo-nucleotide sequences have sufficient complementarity and have thenatural tendency to interact with each other to form a double strandedmolecule. Two nucleotide sequences can form stable interactions witheach other within a wide range of sequence complementarities. Nucleotidesequences with high degrees of complementarity are generally strongerand/or more stable than ones with low degrees of complementarity.Different strengths of interactions may be required for differentprocesses. For example, the strength of interaction for the purpose offorming a stable nucleotide sequence duplex in vitro may be differentfrom that for the purpose of forming a stable interaction between asiRNA and a binding sequence in vivo. The strength of interaction can bereadily determined experimentally or predicted with appropriate softwareby a person skilled in the art.

The terms “hybridize” or “hybridization,” as used herein, refer to theability of a nucleic acid sequence or molecule to base pair with acomplementary sequence and form a duplex nucleic acid structure.Hybridization can be used to test whether two polynucleotides aresubstantially complementary to each other and to measure how stable theinteraction is.

Polynucleotides that share a sufficient degree of complementarity willhybridize to each other under various hybridization conditions.Consequently, polynucleotides that share a high degree ofcomplementarity thus form strong stable interactions and will hybridizeto each other under stringent hybridization conditions. “Stringenthybridization conditions” are well known in the art, as described inSambrook et al. (1989) Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Anexemplary stringent hybridization condition comprises hybridization in6× sodium chloride/sodium citrate (SSC) at about 45° C., followed by oneor more washes in 0.2×SSC and 0.1% SDS at 50-65° C.

“Homologous,” “homology,” “identical,” and “identity,” as used herein,refers to comparisons among nucleic acid sequences. When referring tonucleic acid molecules, “homology,” “similarity,” “identity,” or“percent identity,” refers to the percentage of the nucleotides of aparticular nucleic acid sequence that have been matched to similar oridentical nucleotide sequences by a sequence analysis program. Sequence“identity” or “similarity,” as known in the art, is the relationshipbetween two or more polynucleotide sequences, as determined by comparingthe sequences. In the art, identity also means the degree of sequencerelatedness between polynucleotide sequences, as determined by the matchbetween such sequences. To determine the percent identity or similarityof two nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (i.e., gaps can be introduced in the sequence of afirst nucleic acid sequence for optimal alignment with a second nucleicacid sequence). The nucleotides at corresponding nucleotide positionsare then compared. When a position in the first sequence is occupied bythe same or similar nucleotide as the corresponding position in thesecond sequence, then the molecules are identical or similar at thatposition, respectively. The percent identity or similarity between thetwo sequences is a function of the number of identical or similarpositions shared by the sequences (i.e., the percentage (%) identity isnumber of identical positions divided by the total number of positions(e.g., overlapping positions)×100). Two sequences that share 100%sequence identity are identical. Two sequences that share less than 100%identity, but greater than 50% identity, are similar. Sequences withless than 50% identity are dissimilar.

Both identity and similarity can be readily calculated. Methods commonlyemployed to determine identity or similarity between sequences include,but are not limited to those disclosed in Carillo et al. (1988) SIAM J.Applied Math. 48, 1073. A non-limiting example of a mathematicalalgorithm utilized for the comparison of two sequences is the algorithmof Karlin et al. (1990) Proc. Natl. Acad. Sci. USA 87, 2264-2268,modified as in Karlin et al. (1993) Proc. Natl. Acad. Sci. USA 90,5873-5877. Such an algorithm is incorporated into the NBLAST and XBLASTprograms of Altschul et al. (1990) J. Mol. Biol. 215, 403-410. To obtaingapped alignments for comparison purposes, Gapped BLAST can be utilizedas described in Altschul et al. (1997) Nucleic Acids Res., 25:3389-3402. Alternatively, PSI-Blast can be used to perform an iteratedsearch that detects distant relationships between molecules. Whenutilizing BLAST, Gapped BLAST, and PSI-Blast programs, the defaultparameters of the respective programs (e.g., XBLAST and NBLAST) can beused. Additionally, the FASTA method can also be used. See Altschul etal. (1990) J. Mol. Biol. 215, 403-410. Another example of a mathematicalalgorithm useful for the comparison of sequences is the algorithm ofMyers et al. (1988) CABIOS 4, 11-17. The percent identity between twosequences can also determined using the algorithm of Needleman andWunsch (1970) J. Mol. Biol. 48, 443-453. Another algorithm forcalculating the percent identity between two sequences is determinedusing the local homology method. Smith and Waterman (1981) J. Mol.Biol., 147, 195-197. Optimal alignments may be produced by insertinggaps to maximize the number of matches.

The invention provides methods for attenuating or inhibiting geneexpression in a cell using small interfering RNA (siRNA). The siRNAcontains a nucleotide sequence that hybridizes under physiologicconditions of the cell to the nucleotide sequence of at least a portionof the target mRNA of the gene to be inhibited (i.e., the target gene).The methods described herein do not require 100% sequence identity orcomplementarity between the siRNA and the target gene. By utilizingbioinformatic tools, the sequence can contain mismatching pairs ofnucleotides. Thus, the methods of the invention have the advantage ofbeing able to tolerate some sequence variations that might be expecteddue to genetic mutation, strain polymorphism, or evolutionarydivergence.

Without being bound by theory, it is believed that plants transformedaccording to the invention transcribe an RNA molecule(s) with a regionhomologous to and a region complementary to the pest target gene, andwherein the transcript(s) form a double stranded RNA molecule (dsRNA).The plant recognizes the dsRNA as a potential foreign substance (e.g., asubstance of viral origin). The dicer enzyme of the plant cuts thedouble stranded RNA into pieces of single-stranded RNA of about 23nucleotides in length, called small interfering RNAs (siRNAs). ThesesiRNAs are consumed by invading pests that have entered the plant viathe digestion of plant cells (e.g., cutin). Once absorbed, the siRNAscan be incorporated into the pest's RNA-induced silencing complexes. TheRISC complex can then digest the mRNA of the pest's homologous genelimiting the pest's ability to harm the plant.

siRNA Libraries

A plurality of the above-described siRNA molecules, i.e., two or moresiRNAs, may be used to prepare a siRNA library. Based upon thetarget-specific design of the siRNA molecules, such libraries provide anefficient means for screening for desirable phenotypes in a targetorganism.

Thus, in one aspect of the invention, a method of preparing a library ofsmall interfering RNAs (siRNAs) comprises synthesizing a plurality ofRNA molecules, wherein each RNA molecule comprises (a) a seed sequencecomprising random nucleotides, and (b) a non-seed sequence comprisingdesignated nucleotides. In another aspect of the invention, a method ofpreparing a library of small interfering RNAs (siRNAs) comprisessynthesizing a plurality of RNA molecules, wherein each RNA moleculecomprises (a) a seed sequence comprising nucleotides representative ofone or more microRNA seed sequences of a target organism or an organismrelated to the target organism, and (b) a non-seed sequence comprisingdesignated nucleotides.

According to the disclosed methods, libraries may be prepared by actualsynthesis of each of the plurality of the siRNA molecules, which can beaccomplished using techniques as known in the art, including automatedchemical synthesis, optionally using a mixture of nucleotides to createa randomized sequence. The invention also encompasses in silicopreparation of a library, i.e., using computational techniques togenerate sequences of each of the plurality of siRNA molecules. In manyinstances, in silico library preparation will be useful for initialsteps in library preparation, including steps for exclusion of siRNAmolecules to thereby enrich for target-specific sequences, as describedfurther below.

In one aspect of the invention, a siRNA library comprises siRNAmolecules, wherein each molecule contains a randomized seed sequence,i.e., every possible combination of the four standard ribonucleosides(i.e., adenosine, cytidine, guanosine, and uridine) is stochasticallyrepresented at each position within the randomized seed sequence. Inother aspects of the invention, preparation of a siRNA library involvesone or more steps to exclude siRNA molecules with low complexity and/orlow specificity.

For example, in some aspects of the invention, a method of preparing alibrary can further comprise (a) excluding RNA molecules comprising oneor more nucleotides within the seed sequence, which occur at lowfrequency at corresponding positions of microRNA sequences of a targetorganism or an organism related to a target organism; (b) excluding RNAmolecules comprising a homonucleotide quadruplet within the seedsequence; and/or (c) excluding RNA molecules comprising a seed sequencehaving greater GC content at positions 1-9 than the GC content of thenon-seed sequence at positions 11-19.

The description of nucleotides that occur at low frequency in miRNAs ofa target organism refers to an observed incidence of less than about 50%among a population of naturally occurring miRNAs, or less than about45%, or less than about 40%, or less than about 35%, or less than about30%, or less than about 25%, or less than about 20%, or less than about15%, or less than about 10%, or less than about 5%.

Alternatively stated, siRNAs of the library may be maintained if thesiRNA contains nucleotides within the seed sequence that are observed ata threshold level among naturally occurring miRNAs. Such threshold levelmay be varied as desired, with a higher threshold being generallycorrelated with increased target specificity. For example, a thresholdlevel may be at least about 1%, or greater frequency at which anucleotide is observed among naturally occurring miRNAs, or about 5% orgreater, or about 10% or greater, or about 20% or greater, or about 30%or greater, or about 40% or greater, or about 50% or greater, or about60% or greater, or about 70% or greater, or about 80% or greater, orabout 90% or greater, or about 95% or greater. In a particular aspect ofthe invention, the frequency threshold refers to a nucleotide thatoccurs at a corresponding position in a seed sequence greater than about20% compared to a consensus of seed sequences surveyed from the C.elegans genome. See Example 1. In another aspect of the invention, thefrequency threshold refers to a nucleotide that occurs at acorresponding position in a seed sequence greater than about 20%compared to a consensus of seed sequences surveyed from the Triboliumcastaneum genome. See Example 6.

A “homonucleotide quadruplet” refers to the same nucleotide beingrepeated four times in succession, such as AAAA, within a seed sequence.

The “GC-content” (or guanosine-cytidine content) of a sequence refers tothe percentage of bases in a nucleic acid molecule or sequence orspecific region of a sequence that are either guanosine or cytidine. Forexample, when a seed sequence has at least about 60%, at least about50%, or at least about 40%, or at least about 30%, or at least about20%, or at least about 10%, or at least about 5%, or at least about 1%GC-content and likewise, the non-seed sequence has at most about 40%, atmost about 50%, or at most about 60%, or at most about 70%, or at mostabout 80%, or at most about 90%, or at most about 95%, or at most about99% GC-content, respectively.

Alternatively or in addition, a method of preparing a library canfurther comprise a step of excluding RNA molecules complementary to ahost nucleic acid. In this way, the siRNA molecules of the library willnot include siRNAs likely to be functional for gene silencing in a hostorganism.

A “host” is an organism that is intended for expression or production ofa siRNA. In one aspect of the invention, a host organism is the same asa target organism, i.e., the siRNA is expressed or produced in the sameorganism in which it is intended to be functional. In another aspect ofthe invention, the host organism serves as a carrier of the siRNA to atarget organism. As one example, a host organism can comprise a plant,wherein the target organism is a pest or pathogen of the plant. Inparticular aspects of the invention, the host organism is Glycine max.

In other aspects of the invention, the host organism is Zea mays.

siRNA libraries of the invention include siRNA molecules as describedherein. Accordingly, the seed and non-seed sequences of siRNA moleculeswithin the library can comprise the target-specific seed and non-seedsequences, including consensus sequences, as described herein above. Thescope of the invention is not limited to a library comprising siRNAs forwhich specific sequences are disclosed herein. Rather, sequences fromany organism, both known and presently unknown, can be used to preparetarget-specific siRNAs according to the disclosed methods, as describedfurther herein below.

Additional Compositions Comprising siRNAs

The invention also provides nucleic acids comprising the disclosedsiRNAs, artificial miRNAs, and siRNA libraries. Such nucleic acids aregenerally useful for production or expression of the siRNAs in a mannerthat they can contact target nucleic acids, i.e., nucleic acids to beregulated by the siRNA.

In the context of the invention, the phrase “nucleic acid” or term“nucleotides” refers to oligonucleotides and polynucleotides such asribonucleic acid (RNA) and deoxyribonucleic acid (DNA). The phrasenucleic acid should also be understood to include, as applicable,single-stranded (such as sense or antisense) and double-strandedpolynucleotides. Nucleic acids according to the invention may bepartially or wholly synthetic, and may be isolated and/or purified(i.e., from their natural environment), in substantially pure orhomogeneous form, or free or substantially free of other nucleic acid.

Representative nucleic acids comprising siRNAs of the invention includeexpression constructs and vectors. The term “expression construct”refers to a nucleic acid suitable for expression or production in acell. The term “vector” refers to a nucleic acid molecule (plasmid,virus, bacteriophage, artificial, heterologous, or cut DNA molecule)that can be used to deliver a heterologous or natural polynucleotide ofthe invention into a host cell.

Vectors are capable of being replicated and contain cloning sites forintroduction of a foreign polynucleotide.

Those skilled in the art are readily able to prepare expressionconstructs and vectors of the invention disclosed herein andrecombinantly express the same. For further details see, e.g., Sambrooket al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Such applicabletechniques and protocols for manipulation of nucleic acid, for examplein preparation of nucleic acid constructs, mutagenesis, sequencing,introduction of DNA into cells and gene expression, and analysis ofproteins, are described in detail in Protocols in Molecular Biology,Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992.

Specific expression techniques and vectors previously used with widesuccess upon plants are described by Bevan, Nucl. Acids Res. (1984) 12,8711-8721, and Guerineau and Mullineaux, (1993) “Plant transformationand expression vectors,” Plant Molecular Biology Labfax (Croy RRD, ed.)Oxford, BIOS Scientific Publishers, 121-148.

Expression constructs include a promoter operably linked to a nucleicacid comprising a siRNA, for example, an artificial microRNA, asdescribed herein above. Useful promoters include constitutive promoters,promoters that direct spatially and temporally regulated expression(e.g., tissue-specific and developmental stage-specific promoters), andinducible promoters. Expression constructs may also include enhancers ofgene expression as known in the art.

Tissue-preferred promoters can be utilized to target enhanced expressionof a sequence of interest within a particular plant tissue.Tissue-preferred promoters include Yamamoto et al. (1997) Plant J. 12,255-265; Kawamata et al. (1997) Plant Cell Physiol. 38, 792-803; Hansenet al. (1997) Mol. Gen. Genet. 254, 337-343; Russell et al. (1997)Transgenic Res. 6, 157-168; Rinehart et al. (1996) Plant Physiol. 112,1331-1341; Van Camp et al. (1996) Plant Physiol. 112, 525-535;Canevascini et al. (1996) Plant Physiol. 112, 513-524; Yamamoto et al.(1994) Plant Cell Physiol. 35, 773-778; Lam (1994) Results Probl. CellDiffer. 20, 181-196; Orozco et al. (1993) Plant Mol. Biol. 23,1129-1138; Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA 90,9586-9590; and Guevara-Garcia et al. (1993) Plant J. 4, 495-505. Suchpromoters can be modified, if necessary, for weak expression.

Leaf-preferred promoters are known in the art. See, e.g., Yamamoto etal. (1997) Plant J. 12, 255-265; Kwon et al. (1994) Plant Physiol. 105,357-67; Yamamoto et al. (1994) Plant Cell Physiol. 35, 773-778; Gotor etal. (1993) Plant J. 3, 509-18; Orozco et al. (1993) Plant Mol. Biol. 23,1129-1138; and Matsuoka et al. (1993) Proc. Natl. Acad. Sci. USA 90,9586-9590. In addition, the promoters of cab and rubisco can also beused. See, e.g., Simpson et al. (1958) EMBO J. 4, 2723-2729 and Timko etal. (1988) Nature 318, 57-58.

Root-preferred promoters are known and can be selected from the manyavailable from the literature or isolated de novo from variouscompatible species. See, e.g., Hire et al. (1992) Plant Mol. Biol. 20,207-218 (soybean root-specific glutamine synthetase gene); Keller andBaumgartner (1991) Plant Cell 3, 1051-1061 (root specific controlelement in the GRP 1.8 gene of French bean); Sanger et al. (1990) PlantMol. Biol. 14, 433-443 (root specific promoter of the mannopine synthase(MAS) gene of Agrobacterium tumefaciens); and Miao et al. (1991) PlantCell 3, 11-22 (i.e., a full-length cDNA clone encoding cytosolicglutamine synthetase (GS), which is expressed in roots and root nodulesof soybean). See also Bogusz et al. (1990) Plant Cell 2, 633-641, wheretwo root-specific promoters isolated from hemoglobin genes from thenitrogen-fixing non legume Parasponia andersonii and the relatednon-nitrogen fixing non legume Trema tomentosa are described. Thepromoters of these genes were linked to a 13-glucuronidase reporter geneand introduced into both the non-legume Nicotiana tabacum and the legumeLotus corniculatus, and in both instances root-specific promoteractivity was preserved. Leach and Aoyagi (1991) describe their analysisof the promoters of the highly expressed rolC and rolD root-inducinggenes of Agrobacterium rhizogenes. See Plant Science (Limerick) 79,69-76. They concluded that enhancer and tissue-preferred DNAdeterminants are dissociated in those promoters. Teen et al. (1989) usedgene fusion to lacZ to show that the Agrobacterium T-DNA gene encodingoctopine synthase is especially active in the epidermis of the root tipand that the TR2′ gene is root specific in the intact plant andstimulated by wounding in leaf tissue, an especially desirablecombination of characteristics for use with an insecticidal orlarvicidal gene. See EMBO J. 8 343-350. The TR1′ gene, fused to nptII(neomycin phosphotransferase II) showed similar characteristics.Additional root-preferred promoters include the VfENOD-GRP3 genepromoter and rolB promoter. See also Kuster et al. (1995) Plant Mol.Biol. 29, 759-772; Capana et al. (1994) Plant Mol. Biol. 25, 681-691.See also U.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363; 5,459, 252;5,401,836; 5,110,732; and 5,023,179. The phaseolin gene is described byMurai et al. (1983) Science 23, 476-482, and Sengopta-Gopalen et al.(1988) Proc. Natl. Acad. Sci. USA 82, 3320-3324.

In some aspects, it will be beneficial to express siRNAs of theinvention using an inducible promoter, such as from a pest orpathogen-inducible promoter. Such promoters include those frompathogenesis-related proteins (PR proteins), which are induced followinginfection by a pathogen; e.g., PR proteins, SAR proteins,β-1,3-glucanase, chitinase, etc. See, e.g., Redolfi et al. (1983) Neth.J. Plant Pathol. 89:245-254; Uknes et al. (1992) Plant Cell 4, 645-656;and Van Loon (1985) Plant Mol. Virol. 4, 111-116. See also PCTInternational Publication No. WO 99/43819.

Promoters that are expressed locally at or near the site of pestinfection are particularly of interest. See, e.g., Marineau et al.(1987) Plant Mol. Biol. 9, 335-342; Matton et al. (1989) MolecularPlant-Microbe Interactions 2, 325-331; Somsisch et al. (1986) Proc.Natl. Acad. Sci. USA 83, 2427-2430; Somsisch et al. (1988) Mol. Gen.Genet. 2, 93-98; and Yang (1996) Proc. Natl. Acad. Sci. USA 93,14972-14977. See also, Chen et al. (1996) Plant J. 10, 955-966; Zhang etal. (1994) Proc. Natl. Acad. Sci. USA 91, 2507-2511; Warner et al.(1993) Plant J. 3, 191-201; Siebertz et al. (1989) Plant Cell 1,961-968; U.S. Pat. No. 5,750,386 (nematode-inducible); Cordero et al.(1992) Physiol. Mol. Plant. Path. 41, 189-200, and the references citedtherein.

Additionally, as pests or pathogens enter host plants through wounds orinsect damage, a wound-inducible promoter may be used in the constructsof the invention. Such wound-inducible promoters include potatoproteinase inhibitor (pin /I) gene (Ryan (1990) Ann. Rev. Phytopath. 28,425-449; Duan et al. (1996) Nature Biotech. 14, 494-498); wun1 and wun2,U.S. Pat. No. 5,428,148; winl and wing (Stanford et al. (1989) Mol. Gen.Genet. 215, 200-208); systemin (McGurl et al. (1992) Science 225,1570-1573); WIP1 (Rohmeier et al. (1993) Plant Mol. Biol. 22, 783-792;Eckelkamp et al. (1993) FEBS Lett. 323, 73-76); and the MPI gene(Corderok et al. (1994) Plant J. 6, 141-150). Accumulation ofmetallocarboxypeptidase-inhibitor protein has been reported in leaves ofwounded potato plants (Graham et al. (1981) Biochem. Biophys. Res. Comm.101, 1164-1170). Other studies have focused on genes inducibly regulatedin response to environmental stress or stimuli such as increasedsalinity, drought, and pathogen wounding (Graham et al. (1985) J. Biol.Chem. 260, 6555-6560; Graham et al. (1985) J. Biol. Chem. 260,6561-6564; Smith et al. (1986) Planta 168, 94-100). Other plant genescan be induced by methyl jasmonate, elicitors, heat-shock, anaerobicstress, or herbicide safeners.

U.S. Pat. Nos. 5,589,622 and 5,824,876 describe the identification ofplant genes expressed specifically in or adjacent to the feeding site ofthe plant after attachment by a nematode. The promoters of these planttarget genes can then be used to direct the specific expression ofdetrimental amiRNA to the pest target gene.

In addition to the above-identified promoters, U.S. Patent ApplicationPublication Numbers 2004/0016025, 2007/0056055, 2008/0120750,2009/0183283, and U.S. Pat. Nos. 7,550,578 and 7,615,624 describe avariety of promoters from Oryza sativa and Arabidopsis thaliana, whichmay also be used for expression of siRNAs as described herein. Theparticular promoter sequences of the just named patent documents, anddisclosure regarding use of such promoters, are incorporated byreference herein.

Chemical-regulated promoters can be used to modulate the expression of agene in a plant through the application of an exogenous chemicalregulator. Depending upon the objective, the promoter may be achemical-inducible promoter, where application of the chemical inducesgene expression, or a chemical-repressible promoter, where applicationof the chemical represses gene expression. Chemical inducible promotersare known in the art and include, but are not limited to, the maizeIn2-2 promoter, which is activated by benzenesulfonamide herbicidesafeners, the maize GST promoter, which is activated by hydrophobicelectrophilic compounds that are used as pre-emergent herbicides, andthe tobacco PR-1 a promoter, which is activated by salicylic acid. Otherchemical-regulated promoters of interest include steroidsteroid-responsive promoters (see, e.g., the glucocorticoid-induciblepromoter in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88,10421-10425 and McNellis et al. (1998) Plant J. 14, 247-257) andtetracycline-inducible and tetracycline-repressible promoters (see,e.g., Gatz et al. (1991) Mol. Gen. Genet. 227, 229-237, and U.S. Pat.Nos. 5,814,618 and 5,789,156.

Some suitable promoters initiate transcription only, or predominantly,in certain cell types. Thus, as used herein a cell type- ortissue-preferential promoter is one that drives expressionpreferentially in the target tissue, but may also lead to someexpression in other cell types or tissues as well. It is understood thatsome promoters that show preferential targeting of expression in targettissues may also exhibit “leaky” expression in non-preferential targetedtissues. One example may be a promoter whose expression profile showspreferential expression in maize seed however also exhibits strongexpression in mature leaf tissue. Methods for identifying andcharacterizing promoter regions in plant genomic DNA include, forexample, those described in the following references: Jordano et al.(1989) Plant Cell 1, 855-866; Bustos et al. (1989) Plant Cell 1,839-854; Green et al. (1988) EMBO J. 7, 4035-4044; Meier et al. (1991)Plant Cell 3, 309-316; and Zhang et al. (1996) Plant Physiol. 110,1069-1079.

Promoters active in photosynthetic tissue in order to drivetranscription in green tissues such as leaves and stems are ofparticular interest for the present invention. Most suitable arepromoters that drive expression only or predominantly in such tissues.The promoter may confer expression constitutively throughout the plant,or differentially with respect to the green tissues, or differentiallywith respect to the developmental stage of the green tissue in whichexpression occurs, or in response to external stimuli. Examples of suchpromoters include the ribulose-1,5-bisphosphate carboxylase (RbcS)promoters such as the RbcS promoter from eastern larch (Larix laricina),the pine cab6 promoter (Yamamoto et al. (1994) Plant Cell Physiol. 35,773-778), the Cab-1 gene promoter from wheat (Fejes et al. (1990) PlantMol. Biol. 15, 921-932), the Cab-1 promoter from spinach (Lubberstedt etal. (1994) Plant Physiol. 104, 997-1006), the cab1R promoter from rice(Luan et al. (1992) Plant Cell 4, 971-981), the pyruvate orthophosphatedikinase (PPDK) promoter from maize (Matsuoka et al. (1993) Proc. Natl.Acad. Sci. USA 90, 9586-9590), the tobacco Lhcb1*2 promoter (Cerdan etal. (1997) Plant Mol. Biol. 33, 245-255), the Arabidopsis thaliana SUC2sucrose-H⁺ symporter promoter (Truernit et al. (1995) Planta 196,564-570), and thylakoid membrane protein promoters from spinach (psaD,psaF, psaE, PC, FNR, atpC, atpD, cab, rbcS. Other promoters that drivetranscription in stems, leafs, and green tissue are described in U.S.Patent Publication No. 2007/0006346.

Any of the above-described promoters, or other known promoters, may beused to express siRNAs of the invention. One skilled in the art isreadily able to select a promoter as appropriate for a particularapplication.

The expression constructs and vectors of the invention may be used toprepare compositions for conferring traits to a target or host organism,as described herein below. In one aspect of the invention, such acomposition is a nematicidal composition comprising a siRNA comprisingnucleotides 2-8 of any one of SEQ ID NOs: 1-14, for example, a siRNAcomprising the sequence set forth SEQ ID NOs: 1-14. Compositions forconferring traits may also include two or more siRNAs.

Target Organisms

A target organism is an organism in which siRNAs of the invention areintended to be functional, i.e., to mediate gene silencing orsuppression. In one aspect of the invention, a target organism is also ahost organism, as described herein below. In other aspects of theinvention, a target organism is separate and distinct from a hostorganism that serves as a source of the siRNA to be functional in thetarget organism.

The terms “targeting” or “target(s),” as used herein, refer to theability of siRNA molecules to form base pairs with a complementary mRNAmolecule in a particular organism to thereby result in gene silencing orsuppression. Such an organism is referred to as the target organism. A“target nucleic acid” or “target sequence” is a nucleic acid sequence ormolecule from or in a target organism. Target sequence also implies anucleic acid sequence that is selected for suppression and is notlimited to polynucleotides encoding polypeptides. The target sequencetypically comprises a sequence that is substantially or fullycomplementary to the siRNA. The target sequence includes, but is notlimited to, RNA, DNA, or other polynucleotide comprising the targetsequence.

In one aspect of the invention, “target organisms” are plant pests orpathogens whose damage to the plant can be reduced or eliminatedaccording to the invention. Representative plant pests and pathogensinclude insects, nematodes, fungi, bacteria, viruses, and parasiticplants such as striga, dodder, and mistletoe. Insect pests that may betargeted according to the invention include without limitation chewing,sucking, and boring insects that belong, for example, to thenon-limiting Orders Coleoptera, Diptera, Hemiptera, Heteroptera,Homoptera, Hymenoptera, Lepidoptera, and Orthoptera. Non-limitingexamples of such insect pests are shown in Table 1. Non-limitingexamples of nematodes that may be targeted in accordance with theinvention include those set forth in Table 2. Non-limiting examples offungi, mildews, and rusts that may be targeted in accordance with theinvention include those set forth in Table 3. Non-limiting examples ofbacteria are shown in Table 4. Non-limiting examples of plant virusesthat may be targeted are shown in Table 5.

TABLE 1 Target Pests - Insects Lepidoptera Ostrinia nubilalis, Europeancorn borer Helicoverpa zea, corn earworm Spodoptera exigua, beetarmyworm Spodoptera frugiperda, fall armyworm Diatraea grandiosella,Southwestern corn borer Elasmopalpus lignosellus, lesser cornstalk borerPapaipema nebris, stalk borer Pseudaletia unipuncta, common armywormAgrotis ipsilon, black cutworm Striacosta albicosta, Western beancutworm Spodoptera ornithogalli, yellowstriped armyworm Spodopterapraefica, western yellowstriped armyworm Spodoptera eridania, southernarmyworm Spodoptera eridania, southern armyworm Peridroma saucia,variegated cutworm Papaipema nebris, stalk borer Trichoplusia ni,cabbage looper Keiferia lycopersicella, tomato pinworm Manduca sexta,tobacco hornworm Manduca quinquemaculata, tomato hornworm Artogeiarapae, imported cabbageworm Pieris brassicae, cabbage butterflyTrichoplusia ni, cabbage looper Plutella xylostella, diamondback mothSpodoptera exigua, beet armyworm Agrotis segetum, common cutwormPhthorimaea operculella, potato tuberworm Plutella xylostella,diamondback moth Diatraea saccharalis, sugarcane borer Crymodesdevastator, glassy cutworm Feltia ducens, dingy cutworm Agrotisgladiaria, claybacked cutworm Plathypena scabra, Green cloverwormPseudoplusia includes, Soybean looper Anticarsia gemmatalis, Velvetbeancaterpillar Coleoptera Diabrotica barberi, northern corn rootwormDiabrotica undecimpunctata, southern corn rootworm Diabrotica virgifera,Western corn rootworm Sitophilus zeamais, maize weevil Leptinotarsadecemlineata, Colorado potato beetle Epitrix hirtipennis, tobacco fleabeetle Phyllotreta cruciferae, crucifer flea beetle Phyllotreta pusilla,western black flea beetle Anthonomus eugenii, pepper weevil Leptinotarsadecemlineata, Colorado potato beetle Epitrix cucumeris, potato fleabeetle Hemicrepidus memnonius, wireworms Melanpotus spp., wirewormsCeutorhychus assimilis, cabbage seedpod weevil Phyllotreta cruciferae,crucifer flea beetle Melanolus spp., Aeolus mellillus, wireworm Aeolusmancus, wheat wireworm Horistonotus uhlerii, sand wireworm Sphenophorusmaidis, maize billbug Sphenophorus zeae, timothy bilibug Sphenophorusparvulus, bluegrass billbug Sphenophorus callosus, southern corn billbugPhyllophaga spp., white grubs Chaetocnema pulicaria, corn flea beetlePopillia japonica, Japanese beetle Epilachna varivestis, Mexican beanbeetle Cerotoma trifurcate, Bean leaf beetle Epicauta pestifera,Epicauta lemniscata, Blister beetles Homoptera Rhopalosiphum maidis,corn leaf aphid Anuraphis maidiradicis, corn root aphid Myzus persicae,green peach aphid Macrosiphum euphorbiae, potato aphid Trileurodesvaporariorum, greenhouse whitefly Bemisia tabaci, sweetpotato whiteflyBemisia argentifolii, silverleaf whitefly Brevicoryne brassicae, cabbageaphid Myzus persicae, green peach aphid Empoasca fabae, potatoleafhopper Paratrioza cockerelli, potato psyllid Bemisia argentifolii,silverleaf whitefly Bemisia tabaci, sweetpotato whitefly Cavariellaaegopodii, carrot aphid Brevicoryne brassicae, cabbage aphidSaccharosydne saccharivora, West Indian canefly Sipha flava, yellowsugarcane aphid Spissistilus festinus, Threecornered alfalfa hopperHemiptera Lygus lineolaris, Lygus hesperus, Lygus rugulipennis, lygusbug Acrosternum hilare, green stink bug Euschistus servus, brown stickbug Blissus leucopterus leucopterus, chinch bug Diptera Liriomyzatrifolii, leafminer Liriomyza sativae, vegetable leafminer Scrobipalpulaabsoluta, tomato leafminer Delia platura, seedcorn maggot Deliabrassicae, cabbage maggot Delia radicum, cabbage root fly Psilia rosae,carrot rust fly Tetanops myopaeformis, sugarbeet root maggot OrthopteraMelanoplus differentialis, Differential grasshopper Melanoplusfemurrubrum, Redlegged grasshopper Melanoplus bivittatus, Twostripedgrasshopper

TABLE 2 Target Pests - Parasitic Nematodes Disease Causative Agent AwlDolichoderus spp., D. heterocephalus Bulb and stem (Europe) Ditylenchusdipsaci Burrowing Radopholus similes R. similis Cyst Heterodera avenae,H. zeae, H. schachti; Globodera rostochiensis, G. pallida, and G.tabacum; Heterodera trifolii, H. medicaginis, H. ciceri, H.mediterranea, H. cyperi, H. salixophila, H. zeae, H. goettingiana, H.riparia, H. humuli, H. latipons, H. sorghi, H. fici, H. litoralis, andH. turcomanica; Punctodera chalcoensis Dagger Xiphinema spp., X.americanum, X. Mediterraneum False root-knot Nacobbus dorsalis Lance,Columbia Hoplolaimus Columbus Lance Hoplolaimus spp., H. galeatus LesionPratylenchus spp., P. brachyurus, P. coffeae P. crenatus, P. hexincisus,P. neglectus, P. penetrans, P. scribneri, P. magnica, P. neglectus, P.thornei, P. vulnus, P. zeae Needle Longidorus spp., L. breviannulatusRing Criconemella spp., C. ornata Root-knot Meloidogyne spp., M.arenaria, M. chitwoodi, M. artiellia, M. fallax, M. hapla, M. javanica,M. incognita, M. microtyla, M. partityla, M. panyuensis, M. paranaensisSpiral Helicotylenchus spp. Sting Belonolaimus spp., B. longicaudatusStubby-root Paratrichodorus spp., P. christiei, P. minor, Quinisulciusacutus, Trichodorus spp. Stunt Tylenchorhynchus dubius OthersHirschmanniella species, Pratylenchoid magnicauda

TABLE 3 Target Pathogens - Fungi Disease Causative Agent Brown stripedowny mildew Sclerophthora rayssiae var. zeae Crazy top downy mildewSclerophthora macrospora = S. macrospora Green ear downy mildewSclerospora graminicola Java downy mildew Peronosclerospora maydis =Sclerospora maydis Philippine downy mildew Peronosclerosporaphilippinensis = Sclerospora philippinensis Sorghum downy mildewPeronosclerospora sorghi = Sclerospora sorghi Spontaneum downy mildewPeronosclerospora spontanea = Sclerospora spontanea Sugarcane downymildew Peronosclerospora sacchari = Sclerospora sacchari Dry ear rot(cob, kernel and Nigrospora oryzae (teleomorph: stalk rot) Khuskiaoryzae) Ear rots, minor Aspergillus glaucus, A. niger, Aspergillus spp.,Cunninghamella sp., Curvularia pallescens, Doratomyces stemonitis =Cephalotrichum stemonitis, Fusarium culmorum, Gonatobotrys simplex,Pithomyces maydicus, Rhizopus microsporus, R. stolonifer = R. nigricans,Scopulariopsis brumptii Ergot (horse's tooth, diente Claviceps gigantea(anamorph: Sphacelia sp.) del caballo) Eyespot Aureobasidium zeae =Kabatiella zeae Fusarium ear and stalk rot Fusarium subglutinans = F.moniliforme var. subglutinans Fusarium kernel, root and Fusariummoniliforme (teleomorph: stalk rot, seed rot and Gibberella fujikuroi)seedling blight Fusarium stalk rot, seedling Fusarium avenaceum(teleomorph: root rot Gibberella avenacea) Gibberella ear and stalk rotGibberella zeae (anamorph: Fusarium graminearum) Gray ear rotBotryosphaeria zeae = Physalospora zeae (anamorph: Macrophoma zeae) Grayleaf spot (Cercospora Cercospora sorghi = C. sorghi var. leaf spot)maydis, C. zeae-maydis Helminthosporium root rot Exserohilumpedicellatum = Helminthosporium pedicellatum (teleomorph: Setosphaeria)Hormodendrum ear rot Cladosporium cladosporioides = (Cladosporium rot)Hormodendrum cladosporioides, C. herbarum (teleomorph: Mycosphaerellatassiana) Hyalothyridium leaf spot Hyalothyridium maydis Late wiltCephalosporium maydis Leaf spots, minor Alternaria alternata, Ascochytamaydis, A. tritici, A. zeicola, Bipolaris victoriae = Helminthosporiumvictoriae (teleomorph: Cochliobolus victoriae), C. sativus (anamorph:Bipolaris sorokiniana = H. sorokinianum = H. sativum), Epicoccum nigrum,Exserohilum prolatum = Drechslera prolata (teleomorph: Setosphaeriaprolata) Graphium penicillioides, Leptosphaeria maydis, Leptothyriumzeae, Ophiosphaerella herpotricha, (anamorph: Scolecosporiella sp.),Paraphaeosphaeria michotii, Phoma sp., Septoria zeae, S. zeicola, S.zeina Northern corn leaf blight Exserohilum turcicum = Helminthosporiumturcicum, Setosphaeria turcica Northern corn leaf spot Cochlioboluscarbonum Helminthosporium ear rot Bipolaris zeicola = Helminthosporium(race 1) carbonum Penicillium ear rot (blue Penicillium spp., P.chrysogenum, P. eye, blue mold) expansum, P. oxalicum Phaeocytostromastalk Phaeocytostroma ambiguum, rot and root rot Phaeocytosporella zeaePhaeosphaeria leaf spot Phaeosphaeria maydis, Sphaerulina maydisPhysalospora ear rot Botryosphaeria Botryosphaeria festucae =Physalospora zeicola, (anamorph: Diplodia frumenti) Purple leaf sheathHemiparasitic bacteria and fungi Pyrenochaeta stalk rot Phomaterrestris, Pyrenochaeta terrestris and root rot Pythium root rotPythium spp., P. arrhenomanes, P. graminicola Pythium stalk rot Pythiumaphanidermatum = P. butleri L. Red kernel disease (ear Epicoccum nigrummold, leaf and seed rot) Rhizoctonia ear rot Rhizoctonia zeae(teleomorph: Waitea circinata) Rhizoctonia root rot and Rhizoctoniasolani, Rhizoctonia zeae stalk rot Root rots, minor Alternariaalternata, Cercospora sorghi, Dictochaeta fertilis, Fusarium acuminatum(teleomorph: Gibberella acuminata), F. equiseti (teleomorph: G.intricans), F. oxysporum, F. pallidoroseum, F. poae, F. roseum, F.cyanogena, (anamorph: F. sulphureum), Microdochium bolleyi, Mucor sp.,Periconia circinata, Phytophthora cactorum, P. drechsleri, P. nicotianaevar. parasitica, Rhizopus arrhizus Rostratum leaf spot (leafSetosphaeria rostrata, Helminthosporium disease, ear and stalk rot)(anamorph: Exserohilum rostratum = Helminthosporium rostratum) Rust,common corn Puccinia sorghi Rust, southern corn Puccinia polysora Rust,tropical corn Physopella pallescens, P. zeae = Angiospora zeaeSclerotium ear rot Sclerotium rolfsii (teleomorph: Athelia rolfsii)(southern blight) Seed rot-seedling blight Bipolaris sorokiniana, B.zeicola = Helminthosporium carbonum, Diplodia maydis, Exserohilumpedicellatum, Exserohilum turcicum = Helminthosporium turcicum, Fusariumavenaceum, F. culmorum, F. moniliforme, Gibberella zeae (anamorph: F.graminearum), Macrophomina phaseolina, Penicillium spp., Phomopsis sp.,Pythium spp., Rhizoctonia solani, R. zeae, Sclerotium rolfsii, Spicariasp. Selenophoma leaf spot Selenophoma sp. Sheath rot Gaeumannomycesgraminis Shuck rot Myrothecium gramineum Silage mold Monascus purpureus,M. rubber Smut, common Ustilago zeae = U. maydis Smut, falseUstilaginoidea virens Smut, head Sphacelotheca reiliana = Sporisoriumholci-sorghi Southern corn leaf blight Cochliobolus heterostrophus(anamorph: and stalk rot Bipolaris maydis = Helminthosporium maydis)Southern leaf spot Stenocarpella macrospora = Diplodia macrospora Stalkrots, minor Cercospora sorghi, Fusarium episphaeria, F. merismoides, F.oxysporum, F. poae, F. roseum, F. solani (teleomorph: Nectriahaematococca), F. tricinctum, Mariannaea elegans, Mucor sp.,Rhopographus zeae, Spicaria sp. Storage rots Aspergillus spp.,Penicillium spp. and other fungi Tar spot Phyllachora maydis Trichodermaear rot and Trichoderma viride = T. lignorum root rot (teleomorph:Hypocrea sp.) White ear rot, root and Stenocarpella maydis = Diplodiazeae stalk rot Yellow leaf blight Ascochyta ischaemi, Phyllostictamaydis (teleomorph: Mycosphaerella zeae-maydis) Zonate leaf spotGloeocercospora sorghi Anthracnose leaf blight Colletotrichumgraminicola anthracnose and stalk rot (teleomorph: Glomerellagraminicola), Glomerella tucumanensis (anamorph: Glomerella falcatum)Aspergillus ear and Aspergillus flavus kernel rot Banded leaf and sheathRhizoctonia solani = Rhizoctonia spot microsclerotia (teleomorph:Thanatephorus cucumeris) Black bundle disease Acremonium strictum =Cephalosporium acremonium Black kernel rot Lasiodiplodia theobromae =Botryodiplodia theobromae Borde blanco Marasmiellus sp. Brown spot(black spot, Physoderma maydis stalk rot) Cephalosporium kernelAcremonium strictum = Cephalosporium rot acremonium Charcoal rotMacrophomina phaseolina Corticium ear rot Thanatephorus cucumeris =Corticium sasakii Curvularia leaf spot Curvularia clavata, C.eragrostidis, = C. maculans (teleomorph: Cochliobolus eragrostidis),Curvularia inaequalis, C. intermedia (teleomorph: Cochliobolusintermedius), Curvularia lunata (teleomorph: Cochliobolus lunatus),Curvularia pallescens (teleomorph: Cochliobolus pallescens), Curvulariasenegalensis, C. tuberculata (teleomorph: Cochliobolus tuberculatus)Didymella leaf spot Didymella exitialis Diplodia ear rot and Diplodiafrumenti (teleomorph: stalk rot Botryosphaeria festucae) Diplodia earrot, stalk rot, Diplodia maydis = Stenocarpella maydis seed rot andseedling blight Diplodia leaf spot or leaf Stenocarpella macrospora =Diplodia streak macrospore Corn common rust Puccinia sorghi Cornsouthern rust Puccinia polysora Corn tropical rust Physopellapallescens, P. zeae = Angiospora zeae Oat crown rust Puccinia coronataOat stem Rust Puccinia graminis Stem rust Puccinia graminis = P.graminis f. sp. secalis Leaf (brown) rust Puccinia recondita (anamorph:Aecidium clematitis) Sugarcane common rust Puccinia melanocephala = P.eriantha Wheat leaf (brown) rust Puccinia triticina = P. Recondita f.Sp. tritici = P. tritici-duri Wheat stem (black) rust Puccinia graminis= P. graminis f. sp. tritici Wheat stripe (yellow) rust Pucciniastriiformis (anamorph: P. uredoglumarum) Bean rust Uromycesappendiculatus Cotton rust Puccinia schedonnardi Cotton southwesternrust Puccinia cacabata Cotton tropical rust Phakopsora gossypii Peanutrust Puccinia arachidis Potato common rust Puccinia pittierianap Potatodeforming rust Aecidium cantensis Soybean rust Phakopsora pachyrhizi

TABLE 4 Target Pathogens - Bacteria Disease Causative Agent Bacterialleaf blight and Pseudomonas avenae subsp. avenae stalk rot Bacterialleaf spot Xanthomonas campestris pv. holcicola Bacterial stalk rotEnterobacter dissolvens = Erwinia dissolvens Bacterial stalk and top rotErwinia carotovora subsp. carotovora, Erwinia chrysanthemi pv. ZeaeBacterial stripe Pseudomonas andropogonis Chocolate spot Pseudomonassyringae pv. Coronafaciens Goss's bacterial wilt blight Clavibactermichiganensis subsp. (leaf freckles and wilt) nebraskensis =Cornebacterium michiganense pv. Nebraskense Holcus spot Pseudomonassyringae pv. Syringae Purple leaf sheath Hemiparasitic bacteria + (SeeTable 3) Seed rot-seedling blight Bacillus subtilis Stewart's diseasePantoea stewartii = Erwinia stewartii (bacterial wilt) Corn stunt (MesaCentral or Achapparramiento, stunt, Rio Grande stunt) Spiroplasmakunkelii

TABLE 5 Target Pests - Viruses Alfamoviruses: Alfalfa mosaic alfamovirusBromoviridae Alphacrypto- Alfalfa 1 alphacryptovirus, Beet 1alphacryptovirus, viruses: Beet 2 alphacryptovirus, Beet 3alphacryptovirus, Partitiviridae Carnation 1 alphacryptovirus, Carrottemperate 1 alphacryptovirus, Carrot temperate 3 alphacryptovirus,Carrot temperate 4 alphacryptovirus, Cocksfoot alphacryptovirus, Hoptrefoil 1 alphacryptovirus, Hop trefoil 3 alphacryptovirus, Radishyellow edge alphacryptovirus, Ryegrass alphacryptovirus, Spinachtemperate alphacryptovirus, Vicia alphacryptovirus, White clover 1alphacryptovirus, White clover 3 alphacryptovirus Badnaviruses Bananastreak badnavirus, Cacao swollen shoot badnavirus, Canna yellow mottlebadnavirus, Commelina yellow mottle badnavirus, Dioscorea bacilliformbadnavirus, Kalanchoe top-spotting badnavirus, Rice tungro bacilliformbadnavirus, Schefflera ringspot badnavirus, Sugarcane bacilliformbadnavirus Betacryptoviruses: Carrot temperate 2 betacryptovirus, Hoptrefoil 2 Partitiviridae betacryptovirus, Red clover 2 betacryptovirus,White clover 2 betacryptovirus Bigeminiviruses: Abutilon mosaicbigeminivirus, Ageratum yellow vein Geminiviridae bigeminivirus, Beancalico mosaic bigeminivirus, Bean golden mosaic bigeminivirus, Bhendiyellow vein mosaic bigeminivirus, Cassava African mosaic bigeminivirus,Cassava Indian mosaic bigeminivirus, Chino del tomate bigeminivirus,Cotton leaf crumple bigeminivirus, Cotton leaf curl bigeminivirus,Croton yellow vein mosaic bigeminivirus, Dolichos yellow mosaicbigeminivirus, Euphorbia mosaic bigeminivirus, Horsegram yellow mosaicbigeminivirus, Jatropha mosaic bigeminivirus, Lima bean golden mosaicbigeminivirus, Melon leaf curl bigeminivirus, Mung bean yellow mosaicbigeminivirus, Okra leaf-curl bigeminivirus, Pepper haustecobigeminivirus, Pepper Texas bigeminivirus, Potato yellow mosaicbigeminivirus, Rhynchosia mosaic bigeminivirus, Serrano golden mosaicbigeminivirus, Squash leaf curl bigeminivirus, Tobacco leaf curlbigeminivirus, Tomato Australian leafcurl bigeminivirus, Tomato goldenmosaic bigeminivirus, Tomato Indian leafcurl bigeminivirus, Tomato leafcrumple bigeminivirus, Tomato mottle bigeminivirus, Tomato yellow leafcurl bigeminivirus, Tomato yellow mosaic bigeminivirus, Watermelonchlorotic stunt bigeminivirus, Watermelon curly mottle bigeminivirusBromoviruses: Broad bean mottle bromovirus, Brome mosaic Bromoviridaebromovirus, Cassia yellow blotch bromovirus, Cowpea chlorotic mottlebromovirus, Melandrium yellow fleck bromovirus, Spring beauty latentbromovirus Bymoviruses: Barley mild mosaic bymovirus, Barley yellowmosaic Potyviridae bymovirus, Oat mosaic bymovirus, Rice necrosis mosaicbymovirus, Wheat spindle streak mosaic bymovirus, Wheat yellow mosaicbymovirus Capilloviruses Apple stem grooving capillovirus, Cherry Acapillovirus, Citrus tatter leaf capillovirus, Lilac chlorotic leafspotcapillovirus Carlaviruses Blueberry scorch carlavirus, Cactus 2carlavirus, Caper latent carlavirus, Carnation latent carlavirus,Chrysanthemum B carlavirus, Dandelion latent carlavirus, Elderberrycarlavirus, Fig S carlavirus, Helenium S carlavirus, Honeysuckle latentcarlavirus, Hop American latent carlavirus, Hop latent carlavirus, Hopmosaic carlavirus, Kalanchoe latent carlavirus, Lilac mottle carlavirus,Lily symptomless carlavirus, Mulberry latent carlavirus, Muskmelon veinnecrosis carlavirus, Nerine latent carlavirus, Passiflora latentcarlavirus, Pea streak carlavirus, Poplar mosaic carlavirus, Potato Mcarlavirus, Potato S carlavirus, Red clover vein mosaic carlavirus,Shallot latent carlavirus, Strawberry pseudo mild yellow edge carlavirusCarmoviruses: Bean mild mosaic carmovirus, Cardamine chloroticTombusviridae fleck carmovirus, Carnation mottle carmovirus, Cucumberleaf spot carmovirus, Cucumber soil-borne carmovirus, Galinsoga mosaiccarmovirus, Hibiscus chlorotic ringspot carmovirus, Melon necrotic spotcarmovirus, Pelargonium flower break carmovirus, Turnip crinklecarmovirus Caulimoviruses Blueberry red ringspot caulimovirus, Carnationetched ring caulimovirus, Cauliflower mosaic caulimovirus, Dahlia mosaiccaulimovirus, Figwort mosaic caulimovirus, Horseradish latentcaulimovirus, Mirabilis mosaic caulimovirus, Peanut chlorotic streakcaulimovirus, Soybean chlorotic mottle caulimovirus, Sweet potatocaulimovirus, Thistle mottle caulimovirus Closteroviruses Beet yellowstunt closterovirus, Beet yellows closterovirus, Broad bean severechlorosis closterovirus, Burdock yellows closterovirus, Carnationnecrotic fleck closterovirus, Citrus tristeza closterovirus, Cloveryellows closterovirus, Grapevine stem pitting associated closterovirus,Wheat yellow leaf closterovirus Comoviruses: Bean pod mottle comovirus,Bean rugose mosaic Comoviridae comovirus, Broad bean stain comovirus,Broad bean true mosaic comovirus, Cowpea mosaic comovirus, Cowpea severemosaic comovirus, Glycine mosaic comovirus, Pea mild mosaic comovirus,Potato Andean mottle comovirus, Quail pea mosaic comovirus, Radishmosaic comovirus, Red clover mottle comovirus, Squash mosaic comovirus,Ullucus C comovirus Cucumoviruses: Cucumber mosaic cucumovirus, Peanutstunt Bromoviridae cucumovirus, Tomato aspermy cucumovirus Cytorhabdo-Barley yellow striate mosaic cytorhabdovirus, Broad viruses: bean yellowvein cytorhabdovirus, Broccoli necrotic Rhabdoviridae yellowscytorhabdovirus, Cereal northern mosaic cytorhabdovirus, Festuca leafstreak cytorhabdovirus, Lettuce necrotic yellows cytorhabdovirus,Sonchus cytorhabdovirus, Strawberry crinkle cytorhabdovirusDianthoviruses Carnation ringspot dianthovirus, Red clover necroticmosaic dianthovirus, Sweet clover necrotic mosaic dianthovirusEnamoviruses Pea enation mosaic enamovirus Fijiviruses: Maize roughdwarf fijivirus, Oat sterile dwarf fijivirus, Reoviridae Pangola stuntfijivirus, Rice black-streaked dwarf fijivirus, Sugarcane Fiji diseasefijivirus Furoviruses Beet necrotic yellow vein furovirus, Beetsoil-borne furovirus, Broad bean necrosis furovirus, Oat golden stripefurovirus, Peanut clump furovirus, Potato mop- top furovirus, Sorghumchlorotic spot furovirus, Wheat soil-borne mosaic furovirusHordeiviruses Anthoxanthum latent blanching hordeivirus, Barley stripemosaic hordeivirus, Lychnis ringspot hordeivirus, Poa semilatenthordeivirus Hybrigemini- Beet curly top hybrigeminivirus, Tomato pseudocurly viruses: top hybrigeminivirus Geminiviridae Idaeoviruses Raspberrybushy dwarf idaeovirus Ilarviruses: Apple mosaic ilarvirus, Asparagus 2ilarvirus, Bromoviridae Blueberry necrotic shock ilarvirus, Citrus leafrugose ilarvirus, Citrus variegation ilarvirus, Elm mottle ilarvirus,Humulus japonicus ilarvirus, Hydrangea mosaic ilarvirus, Lilac ringmottle ilarvirus, Parietaria mottle ilarvirus, Plum American linepattern ilarvirus, Prune dwarf ilarvirus, Prunus necrotic ringspotilarvirus, Spinach latent ilarvirus, Tobacco streak ilarvirus, Tulareapple mosaic ilarvirus Ipomoviruses: Sweet potato mild mottleipomovirus, Sweet potato Potyviridae yellow dwarf ipomovirus LuteoviruseBarley yellow dwarf luteovirus, Bean leaf roll luteovirus, Beet mildyellowing luteovirus, Beet western yellows luteovirus, Carrot red leafluteovirus, Groundnut rosette assistor luteovirus, Potato leafrollluteovirus, Solanum yellows luteovirus, Soybean dwarf luteovirus,Soybean Indonesian dwarf luteovirus, Strawberry mild yellow edgeluteovirus, Subterranean clover red leaf luteovirus, Tobacco necroticdwarf luteovirus Machlomoviruses Maize chlorotic mottle machlomovirusMacluraviruse Maclura mosaic macluravirus, Narcissus latent macluravirusMarafiviruses Bermuda grass etched-line marafivirus, Maize rayado finomarafivirus, Oat blue dwarf marafivirus Monogemini- Chloris striatemosaic monogeminivirus, Digitaria viruses: striate mosaicmonogeminivirus, Digitaria streak Geminiviridae monogeminivirus, Maizestreak monogeminivirus, Miscanthus streak monogeminivirus, Panicumstreak monogeminivirus, Paspalum striate mosaic monogeminivirus,Sugarcane streak monogeminivirus, Tobacco yellow dwarf monogeminivirus,Wheat dwarf monogeminivirus Nanaviruses Banana bunchy top nanavirus,Coconut foliar decay nanavirus, Faba bean necrotic yellows nanavirus,Milk vetch dwarf nanavirus, Subterranean clover stunt nanavirusNecroviruses Tobacco necrosis necrovirus, Carnation yellow stripenecrovirus, Lisianthus necrosis necrovirus Nepoviruses: Arabis mosaicnepovirus, Arracacha A nepovirus, Comoviridae Artichoke Italian latentnepovirus, Artichoke yellow ringspot nepovirus, Blueberry leaf mottlenepovirus, Cacao necrosis nepovirus, Cassava green mottle nepovirus,Cherry leaf roll nepovirus, Cherry rasp leaf nepovirus, Chicory yellowmottle nepovirus, Crimson clover latent nepovirus, Cycas necrotic stuntnepovirus, Grapevine Bulgarian latent nepovirus, Grapevine chrome mosaicnepovirus, Grapevine fanleaf nepovirus, Hibiscus latent ringspotnepovirus, Lucerne Australian latent nepovirus, Mulberry ringspotnepovirus, Myrobalan latent ringspot nepovirus, Olive latent ringspotnepovirus, Peach rosette mosaic nepovirus, Potato black ringspotnepovirus, Potato U nepovirus, Raspberry ringspot nepovirus, Tobaccoringspot nepovirus, Tomato black ring nepovirus, Tomato ringspotnepovirus Nucleorhabdo- Carrot latent nucleorhabdovirus, Corianderfeathery viruses: red vein nucleorhabdovirus, Cow parsnip mosaicRhabdoviridae nucleorhabdovirus, Cynodon chlorotic streaknucleorhabdovirus, Datura yellow vein nucleorhabdovirus, Eggplantmottled dwarf nucleorhabdovirus, Maize mosaic nucleorhabdovirus,Pittosporum vein yellowing nucleorhabdovirus, Potato yellow dwarfnucleorhabdovirus, Sonchus yellow net nucleorhabdovirus, Sowthistleyellow vein nucleorhabdovirus, Tomato vein clearing nucleorhabdovirus,Wheat American striate mosaic nucleorhabdovirus Oryzaviruses:Echinochloa ragged stunt oryzavirus, Rice ragged Reoviridae stuntoryzavirus Ourmiaviruse Cassava Ivorian bacilliform ourmiavirus, Epiruscherry ourmiavirus, Melon Ourmia ourmiavirus, Pelargonium zonate spotourmiavirus Phytoreoviruses: Clover wound tumor phytoreovirus, Ricedwarf Reoviridae phytoreovirus, Rice gall dwarf phytoreovirus, Ricebunchy stunt phytoreovirus, Sweet potato phytoreovirus PotexvirusesAsparagus 3 potexvirus, Cactus X potexvirus, Cassava X potexvirus,Chicory X potexvirus, Clover yellow mosaic potexvirus, Commelina Xpotexvirus, Cymbidium mosaic potexvirus, Daphne X potexvirus, Foxtailmosaic potexvirus, Hydrangea ringspot potexvirus, Lily X potexvirus,Narcissus mosaic potexvirus, Nerine X potexvirus, Papaya mosaicpotexvirus, Pepino mosaic potexvirus, Plantago asiatica mosaicpotexvirus, Plantain X potexvirus, Potato aucuba mosaic potexvirus,Potato X potexvirus, Tulip X potexvirus, Viola mottle potexvirus, Whiteclover mosaic potexvirus Potyviruses: Alstroemeria mosaic potyvirus,Amaranthus leaf Potyviridae mottle potyvirus, Araujia mosaic potyvirus,Arracacha Y potyvirus, Artichoke latent potyvirus, Asparagus 1potyvirus, Banana bract mosaic potyvirus, Bean common mosaic necrosispotyvirus, Bean common mosaic potyvirus, Bean yellow mosaic potyvirus,Beet mosaic potyvirus, Bidens mosaic potyvirus, Bidens mottle potyvirus,Cardamom mosaic potyvirus, Carnation vein mottle potyvirus, Carrot thinleaf potyvirus, Cassava brown streak potyvirus, Cassia yellow spotpotyvirus, Celery mosaic potyvirus, Chickpea bushy dwarf potyvirus,Chickpea distortion mosaic potyvirus, Clover yellow vein potyvirus,Commelina diffusa potyvirus, Commelina mosaic potyvirus, Cowpea greenvein-banding potyvirus, Cowpea Moroccan aphid-borne mosaic potyvirus,Cowpea rugose mosaic potyvirus, Crinum mosaic potyvirus, Daphne Ypotyvirus, Dasheen mosaic potyvirus, Datura Colombian potyvirus, Daturadistortion mosaic potyvirus, Datura necrosis potyvirus, Daturashoestring potyvirus, Dendrobium mosaic potyvirus, Desmodium mosaicpotyvirus, Dioscorea alata potyvirus, Dioscorea green banding mosaicpotyvirus, Eggplant green mosaic potyvirus, Euphorbia ringspotpotyvirus, Freesia mosaic potyvirus, Groundnut eyespot potyvirus, Guarsymptomless potyvirus, Guinea grass mosaic potyvirus, Helenium Ypotyvirus, Henbane mosaic potyvirus, Hippeastrum mosaic potyvirus,Hyacinth mosaic potyvirus, Iris fulva mosaic potyvirus, Iris mild mosaicpotyvirus, Iris severe mosaic potyvirus, Johnsongrass mosaic potyvirus,Kennedya Y potyvirus, Leek yellow stripe potyvirus, Lettuce mosaicpotyvirus, Lily mottle potyvirus, Maize dwarf mosaic potyvirus, Malvavein clearing potyvirus, Marigold mottle potyvirus, Narcissus yellowstripe potyvirus, Nerine potyvirus, Onion yellow dwarf potyvirus,Ornithogalum mosaic potyvirus, Papaya ringspot potyvirus, Parsnip mosaicpotyvirus, Passiflora ringspot potyvirus, Passiflora South Africanpotyvirus, Passionfruit woodiness potyvirus, Patchouli mosaic potyvirus,Pea mosaic potyvirus, Pea seed-borne mosaic potyvirus, Peanut greenmosaic potyvirus, Peanut mottle potyvirus, Pepper Indian mottlepotyvirus, Pepper mottle potyvirus, Pepper severe mosaic potyvirus,Pepper veinal mottle potyvirus, Plum pox potyvirus, Pokeweed mosaicpotyvirus, Potato A potyvirus, Potato V potyvirus, Potato Y potyvirus,Primula mosaic potyvirus, Ranunculus mottle potyvirus, Sorghum mosaicpotyvirus, Soybean mosaic potyvirus, Statice Y potyvirus, Sugarcanemosaic potyvirus, Sweet potato feathery mottle potyvirus, Sweet potato Gpotyvirus, Swordbean distortion mosaic potyvirus, Tamarillo mosaicpotyvirus, Telfairia mosaic potyvirus, Tobacco etch potyvirus, Tobaccovein-banding mosaic potyvirus, Tobacco vein mottling potyvirus, Tobaccowilt potyvirus, Tomato Peru potyvirus, Tradescantia- Zebrina potyvirus,Tropaeolum 1 potyvirus, Tropaeolum 2 potyvirus, Tuberose potyvirus,Tulip band-breaking potyvirus, Tulip breaking potyvirus, Tulip chloroticblotch potyvirus, Turnip mosaic potyvirus, Ullucus mosaic potyvirus,Vallota mosaic potyvirus, Vanilla mosaic potyvirus, Vanilla necrosispotyvirus, Voandzeia distortion mosaic potyvirus, Watermelon mosaic 1potyvirus, Watermelon mosaic 2 potyvirus, Wild potato mosaic potyvirus,Wisteria vein mosaic potyvirus, Yam mosaic potyvirus, Zucchini yellowfleck potyvirus, Zucchini yellow mosaic potyvirus Rymoviruses: Hordeummosaic rymovirus, Oat necrotic mottle Potyviridae rymovirus, Ryegrassmosaic rymovirus, Wheat streak Agropyron mosaic rymovirus mosaicrymovirus Satellite RNAs Arabis mosaic satellite RNA, Chicory yellowmottle satellite RNA, Cucumber mosaic satellite RNA, Grapevine fanleafsatellite RNA, Strawberry latent ringspot satellite RNA, Tobaccoringspot satellite RNA, Tomato black ring satellite RNA, Velvet tobaccomottle satellite RNA Satelliviruses Maize white line mosaicsatellivirus, Panicum mosaic satellivirus, Tobacco mosaic satellivirus,Tobacco necrosis satellivirus Sequiviruses: Dandelion yellow mosaicsequivirus, Parsnip yellow Sequiviridae fleck sequivirus SobemovirusesBean southern mosaic sobemovirus, Blueberry shoestring sobemovirus,Cocksfoot mottle sobemovirus, Lucerne transient streak sobemovirus, Riceyellow mottle sobemovirus, Rottboellia yellow mottle sobemovirus,Solanum nodiflorum mottle sobemovirus, Sowbane mosaic sobemovirus,Subterranean clover mottle sobemovirus, Turnip rosette sobemovirus,Velvet tobacco mottle sobemovirus Tenuiviruses Maize stripe tenuivirus,Rice grassy stunt tenuivirus, Rice hoja blanca tenuivirus, Rice stripetenuivirus Tobamoviruses Cucumber green mottle mosaic tobamovirus,Frangipani mosaic tobamovirus, Kyuri green mottle mosaic tobamovirus,Odontoglossum ringspot tobamovirus, Paprika mild mottle tobamovirus,Pepper mild mottle tobamovirus, Ribgrass mosaic tobamovirus, OpuntiaSammons' tobamovirus, Sunn- hemp mosaic tobamovirus, Tobacco mild greenmosaic tobamovirus, Tobacco mosaic tobamovirus, Tomato mosaictobamovirus, Ullucus mild mottle tobamovirus Tobraviruses Pea earlybrowning tobravirus, Pepper ringspot tobravirus, Tobacco rattletobravirus Tombusviruses: Artichoke mottled crinkle tombusvirus,Carnation Tombusviridae Italian ringspot tombusvirus, Cucumber necrosistombusvirus, Cymbidium ringspot tombusvirus, Eggplant mottled crinkletombusvirus, Grapevine Algerian latent tombusvirus, Lato Rivertombusvirus, Neckar River tombusvirus, Pelargonium leaf curltombusvirus, Pepper Moroccan tombusvirus, Petunia asteroid mosaictombusvirus, Tomato bushy stunt tombusvirus Tospoviruses: Impatiensnecrotic spot tospovirus, Peanut yellow Bunyaviridae spot tospovirus,Tomato spotted wilt tospovirus Trichoviruses Apple chlorotic leaf spottrichovirus, Heracleum latent trichovirus, Potato T trichovirusTymoviruses Abelia latent tymovirus, Belladonna mottle tymovirus, Cacaoyellow mosaic tymovirus, Clitoria yellow vein tymovirus, Desmodiumyellow mottle tymovirus, Dulcamara mottle tymovirus, Eggplant mosaictymovirus, Erysimum latent tymovirus, Kennedya yellow mosaic tymovirus,Melon rugose mosaic tymovirus, Okra mosaic tymovirus, Ononis yellowmosaic tymovirus, Passionfruit yellow mosaic tymovirus, Physalis mosaictymovirus, Plantago mottle tymovirus, Potato Andean latent tymovirus,Scrophularia mottle tymovirus, Turnip yellow mosaic tymovirus, Voandzeianecrotic mosaic tymovirus, Wild cucumber mosaic tymovirus UmbravirusesBean yellow vein banding umbravirus, Carrot mottle mimic umbravirus,Carrot mottle umbravirus, Carrot mottle mimic umbravirus, Groundnutrosette umbravirus, Lettuce speckles mottle umbravirus, Tobacco mottleumbravirus Varicosaviruses Freesia leaf necrosis varicosavirus, Lettucebig-vein varicosavirus, Tobacco stunt varicosavirus Waikaviruses:Anthriscus yellows waikavirus, Maize chlorotic dwarf Sequiviridaewaikavirus, Rice tungro spherical waikavirus Putative Alsike clover veinmosaic virus, Alstroemeria streak Ungrouped potyvirus, Amaranthus mosaicpotyvirus, Amazon lily Viruses mosaic potyvirus, Anthoxanthum mosaicpotyvirus, Apple stem pitting virus, Aquilegia potyvirus, Asclepiasrhabdovirus, Atropa belladonna rhabdovirus, Barley mosaic virus, Barleyyellow streak mosaic virus, Beet distortion mosaic virus, Beet leaf curlrhabdovirus, Beet western yellows ST9- associated RNA virus, Blackraspberry necrosis virus, Bramble yellow mosaic potyvirus, Brinjal mildmosaic potyvirus, Broad bean B virus, Broad bean V potyvirus, Broad beanyellow ringspot virus, Bryonia mottle potyvirus, Burdock mosaic virus,Burdock mottle virus, Callistephus chinensis chlorosis rhabdovirus,Canary reed mosaic potyvirus, Canavalia maritima mosaic potyvirus,Carnation rhabdovirus, Carrot mosaic potyvirus, Cassava symptomlessrhabdovirus, Cassia mosaic virus, Cassia ringspot virus, Celery yellowmosaic potyvirus, Celery yellow net virus, Cereal flame chlorosis virus,Chickpea filiform potyvirus, Chilli veinal mottle potyvirus,Chrysanthemum spot potyvirus, Chrysanthemum vein chlorosis rhabdovirus,Citrus leprosis rhabdovirus, Citrus ringspot virus, Clover mild mosaicvirus, Cocksfoot streak potyvirus, Colocasia bobone disease rhabdovirus,Cucumber toad-skin rhabdovirus, Cucumber vein yellowing virus,Cypripedium calceolus potyvirus, Datura innoxia Hungarian mosaicpotyvirus, Dioscorea trifida potyvirus, Dock mottling mosaic potyvirus,Dodonaea yellows-associated virus, Eggplant severe mottle potyvirus,Euonymus fasciation rhabdovirus, Euonymus rhabdovirus, Fern potyvirus,Fig potyvirus, Gerbera symptomless rhabdovirus, Grapevine fleck virus,Grapevine stunt virus, Guar top necrosis virus, Habenaria mosaicpotyvirus, Holcus lanatus yellowing rhabdovirus, Holcus streakpotyvirus, Iris germanica leaf stripe rhabdovirus, Iris Japanesenecrotic ring virus, Isachne mosaic potyvirus, Kalanchoe isometricvirus, Kenaf vein-clearing rhabdovirus, Launaea mosaic potyvirus, Lupinyellow vein rhabdovirus, Maize eyespot virus, Maize line virus, Maizemottle/chlorotic stunt virus, Maize white line mosaic virus, Malvastrummottle virus, Melilotus mosaic potyvirus, Melon vein-banding mosaicpotyvirus, Melothria mottle potyvirus, Mimosa mosaic virus, Mung beanmottle potyvirus, Narcissus degeneration potyvirus, Narcissus lateseason yellows potyvirus, Nerine Y potyvirus, Nothoscordum mosaicpotyvirus, Oak ringspot virus, Orchid fleck rhabdovirus, Palm mosaicpotyvirus, Parsley green mottle potyvirus, Parsley rhabdovirus, Parsnipleafcurl virus, Passionfruit Sri Lankan mottle potyvirus, Passionfruitvein-clearing rhabdovirus, Patchouli mottle rhabdovirus, Pea stemnecrosis virus, Peanut top paralysis potyvirus, Peanut veinal chlorosisrhabdovirus, Pecteilis mosaic potyvirus, Pepper mild mosaic potyvirus,Perilla mottle potyvirus, Pigeonpea proliferation rhabdovirus, Pigeonpeasterility mosaic virus, Plantain 7 potyvirus, Plantain mottlerhabdovirus, Pleioblastus chino potyvirus, Poplar decline potyvirus,Primula mottle potyvirus, Purple granadilla mosaic virus, Ranunculusrepens symptomless rhabdovirus, Rice yellow stunt virus, Saintpaulialeaf necrosis rhabdovirus, Sambucus vein clearing rhabdovirus,Sarracenia purpurea rhabdovirus, Shamrock chlorotic ringspot potyvirus,Soybean mild mosaic virus, Soybean rhabdovirus, Soybean spherical virus,Soybean yellow vein virus, Soybean Z potyvirus, Strawberry latent Crhabdovirus, Strawberry mottle virus, Strawberry pallidosis virus,Sunflower mosaic potyvirus, Sweet potato latent potyvirus, Teasel mosaicpotyvirus, Thimbleberry ringspot virus, Tomato mild mottle potyvirus,Trichosanthes mottle potyvirus, Tulip halo necrosis virus, Tulip mosaicvirus, Turnip vein-clearing virus, Urd bean leaf crinkle virus, Vignasinensis mosaic rhabdovirus, Watercress yellow spot virus, WatermelonMoroccan mosaic potyvirus, Wheat chlorotic spot rhabdovirus, Whitebryony potyvirus, Wineberry latent virus, Zinnia mild mottle potyvirus,Zoysia mosaic potyvirus

A “non-target organism(s),” as used herein, is/are any organism(s) otherthan the target organism(s). Where the target organism and host organismdiffer, a non-target organism can comprise a host organism and organismsthat consume the host organism or otherwise contact siRNAs expressed ina host organism. The target-specific design of siRNAs, as describedherein, provides that such siRNAs have little or no gene silencingactivity in non-target organisms.

Host Organisms

A “host” or “host organism” as used herein refers to an organism thatexpresses or produces siRNA. The host organism may transiently or stablyexpress the siRNA. A host organism may be a transgenic organism. In oneaspect of the invention, a host organism is the same as a targetorganism, i.e., the siRNA is expressed in the same organism in which itis intended to be functional. In another aspect of the invention, thehost organism serves as a carrier of the siRNA to a target organism. Asone non-limiting example, a host organism is a plant, wherein the targetorganism is a pest or pathogen of the plant. In another example, thehost organism may be a food source for a target organism.

A “host nucleic acid” is a nucleic acid from or in a host organism, forexample, a nucleic acid from or in a plant or plant part.

The term “expression,” as used herein with regard to siRNA or miRNArefers to transcription of a siRNA/miRNA nucleotide sequence driven byits promoter. Expression as used herein also includes the production ofsiRNAs or miRNAs from larger RNA transcripts. As such, a host organismmay express a RNA that is processed to produce or express one or moresiRNAs or miRNAs.

Plants useful as host organisms include any of various photosynthetic,eukaryotic, multicellular organisms of the kingdom Plantae, includingboth monocots and dicots. The term “plant” includes reference to wholeplants, plant parts, plant organs, plant tissues, plant cells, seeds,and progeny of the same. Plant cells include, without limitation, cellsfrom seeds, suspension cultures, embryos, meristematic regions, callustissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, andmicrospores. Plant also refers to plants or plant parts that stably ortransiently express a gene product, including a siRNA.

A “plant part” is any portion of a plant regardless of whether it isisolated or attached to an intact plant. The phrase “plant part”includes differentiated and undifferentiated tissues including, but notlimited to the following: roots, stems, shoots, leaves, pollen, seeds,tumor tissue, and various forms of cells and culture (e.g., singlecells, protoplasts, embryos, and callus tissue). The plant tissue may bein plant or in a plant organ, tissue, or cell culture. Plant parts alsoinclude plant products, such as grains, seeds, fruits, and nuts orcommodity products.

A “plant product” refers to an agricultural or commercial productcreated from a plant, plant part, or seed. Non-limiting examples ofplant products include flowers, pollen, leaves, vines, stalks, fruits,vegetables, cucurbits, roots, tubers, cones, pods, seeds, beans, grains,kernels, and hulls.

Some plant products are processed and thus become “commodity products.”As used herein, “commodity products” include, but are not limited to,whole or processed seeds, beans, grains, kernels, hulls, meals, grits,flours, sugars, starches, protein concentrates, protein, lipids,carbohydrates, nucleic acids, metabolites, chlorophylls, waxes, oils,extracts, juices, concentrates, liquids, syrups, feed, silage, fiber,wood, pulp, paper, pigments, natural products, toxins, or other food orproduct produced from plants.

Commodity products containing one or more of the nucleotide sequences ofthe invention, or produced from a transformed plant, recombinant plant,or seed containing one or more of the nucleotide sequences of theinvention are specifically contemplated as aspects of the invention as ameans of identifying or detecting the source of the plant product orcommodity. Such aspects are referred to herein as “biological samples.”The identification or detection of one or more of the nucleotidesequences of the invention in one or more biological samples is de factoevidence that the plant product or commodity product comprises a plantor plant part of the invention disclosed herein.

As used herein, “a nucleotide sequence of the invention” comprises thesiRNAs, miRNAs, or constructs thereof as disclosed herein. Suchnucleotide sequences of the invention can be used to identify plants,plant products, or commodity products containing one or more of thenucleotide sequence of the invention using any number of techniquesknown to those having skill in the art such as through PCR-basedmethods, southern blotting, northern blotting, or microarray analyses.In this particular aspect, the functionality of nucleotide sequence ofthe invention (i.e., siRNA or miRNA) is immaterial and the presence ofthe nucleotide sequence in the plant or plant product serves tospecifically identify or detect the source of the plant part, plantproduct, or commodity.

Representative host plants include soybean (Glycine max), corn (Zeamays), canola (Brassica napus, Brassica rapa ssp.), alfalfa (Medicagosativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghumbicolor, Sorghum vulgare), sunflower (Helianthus annuus), wheat(Triticum aestivum), tobacco (Nicotiana tabacum), potato (Solanumtuberosum), peanuts (Arachis hypogaea), cotton (Gossypium hirsutum),sweet potato (Ipomoea batatas), cassava (Manihot esculenta), coffee(Coffea ssp.), coconut (Cocos nucifera), pineapple (Ananas comosus),citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camelliasinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficuscarica), guava (Psidium guajava), mango (Mangifera indica), olive (Oleaeuropaea), papaya (Carica papaya), cashew (Anacardium occidental),macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugarbeets (Beta vulgaris), oats, barley, vegetables, ornamentals, andconifers.

Additional host plants of the invention are crop plants, for example,cereals and pulses, maize, wheat, potatoes, tapioca, rice, sorghum,millet, cassava, barley, pea, and other root, tuber, or seed crops.Important seed crops for the invention are oil-seed rape, sugar beet,maize, sunflower, soybean, and sorghum. Horticultural plants to whichthe invention may be applied may include lettuce, endive, and vegetablebrassica including cabbage, broccoli, and cauliflower, and carnations,geraniums, petunias, and begonias. The invention may be applied totobacco, cucurbits, carrot, strawberry, sunflower, tomato, pepper,chrysanthemum, poplar, eucalyptus, and pine. Optionally, plants of theinvention include grain seeds, such as corn, wheat, barley, rice,sorghum, rye, etc. Optionally, plants of the invention include oil-seedplants. Oil seed plants include canola, cotton, soybean, safflower,sunflower, brassica, maize, alfalfa, palm, coconut, etc. Optionally,plants of the invention include leguminous plants. Leguminous plantsinclude beans and peas. Beans include guar, locust bean, fenugreek,soybean, garden beans, cowpea, mung bean, lima bean, fava bean, lentils,chickpea, etc. Host plants useful in the invention are row crops andbroadcast crops. Non-limiting examples of useful row crops are corn,soybeans, cotton, amaranth, vegetables, rice, sorghum, wheat, milo,barley, sunflower, durum, and oats. Non-limiting examples of usefulbroadcast crops are sunflower, millet, rice, sorghum, wheat, milo,barley, durum, and oats. Host plants useful in the invention aremonocots and dicots. Non-limiting examples of useful monocots are rice,corn, wheat, palm trees, turf grasses, barley, and oats. Non-limitingexamples of useful dicots are soybean, cotton, alfalfa, canola, flax,tomato, sugar beet, sunflower, potato, tobacco, corn, wheat, rice,lettuce, celery, cucumber, carrot, and cauliflower, grape, and turfgrasses. Host plants useful in the invention include plants cultivatedfor aesthetic or olfactory benefits. Non-limiting examples includeflowering plants, trees, grasses, shade plants, and flowering andnon-flowering ornamental plants. Host plants useful in the inventioninclude plants cultivated for nutritional value, fibers, wood, andindustrial products.

One skilled in the art will recognize the wide variety of host cellsthat can be transformed with the vectors according to the inventiondisclosed herein. Non-limiting examples of such cells are those inembryogenic tissue, callus tissue types I, II, and III, hypocotyl,meristem, root tissue, tissues for expression in phloem, and the like.

Almost all plant tissues may be transformed during dedifferentiationusing appropriate techniques described herein. Recipient cell targetsinclude, but are not limited to, meristem cells, Type I, Type II, andType III callus, immature embryos, and gametic cells such asmicrospores, pollen, sperm, and egg cells. It is contemplated that anycell from which a fertile plant may be regenerated is useful as arecipient cell. Type I, Type II, and Type III callus may be initiatedfrom tissue sources including, but not limited to, immature embryos,immature inflorescences, seedling apical meristems, microspores, and thelike.

Those cells that are capable of proliferating as callus also arerecipient cells for genetic transformation. Techniques for transformingimmature embryos and subsequent regeneration of fertile transgenicplants are well known in the art. Direct transformation of immatureembryos obviates the need for long-term development of recipient cellcultures. Pollen, as well as its precursor cells, microspores, may becapable of functioning as recipient cells for genetic transformation, oras vectors to carry foreign DNA for incorporation during fertilization.Direct pollen transformation obviates the need for cell culture.

Meristematic cells (i.e., plant cells capable of continual cell divisionand characterized by an undifferentiated cytological appearance,normally found at growing points or tissues in plants such as root tips,stem apices, lateral buds, etc.) may represent another type of recipientplant cell. Because of their undifferentiated growth and capacity fororgan differentiation and totipotency, a single transformed meristematiccell could be recovered as a completely transformed plant. In fact, itis proposed that embryogenic suspension cultures may be an in vitromeristematic cell system, retaining ability for continued cell divisionin an undifferentiated state, controlled by the media environment.

Wide varieties of techniques are available for introducing siRNAs of theinvention into a host under conditions that allow for stable maintenanceand expression of the siRNA. The particular choice of a transformationtechnology will be determined by its efficiency to transform certainplant species as well as the experience and preference of the personpracticing the invention with a particular methodology of choice. Itwill be apparent to the skilled person that the particular choice of atransformation system to introduce nucleic acid into plant cells is notessential to or a limitation of the invention, nor is the choice oftechnique for plant regeneration.

Transformation protocols as well as protocols for introducingheterologous nucleic acids into plants may vary depending on the type ofplant or plant cell, i.e., monocot or dicot, targeted fortransformation. Suitable methods of introducing the DNA constructinclude microinjection (Crossway et al. (1986) Biotechniques 4, 320-334;and U.S. Pat. No. 6,300,543); sexual crossing, electroporation (Riggs etal. (1986) Proc. Natl. Acad. Sci. USA 83, 5602-5606);Agrobacterium-mediated transformation (Townsend et al., U.S. Pat. Nos.5,563,055 and 5,981,840); direct gene transfer (Paszkowski et al. (1984)EMBO J. 3, 2717-2722); and ballistic particle acceleration (see, e.g.,Sanford et al., U.S. Pat. No. 4,945,050; Tomes et al., U.S. Pat. No.5,879,918; Tomes et al., U.S. Pat. No. 5,886,244; Bidney et al., U.S.Pat. No. 5,932,782; Tomes et al. (1995) “Direct DNA Transfer into IntactPlant Cells via Microprojectile Bombardment,” in Plant Cell, Tissue, andOrgan Culture: Fundamental Methods, ed. Gamborg and Phillips(Springer-Verlag, Berlin); and McCabe et al. (1988) Biotechnology 6,923-926). See also Weissinger et al. (1988) Ann. Rev. Genet. 22,421-477; Sanford et al. (1987) Particulate Science and Technology 5,27-37 (onion); Christou et al. (1988) Plant Physiol. 87, 671-674(soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P,175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet. 96, 319-324(soybean); Datta et al. (1990) Biotechnology 8, 736-740 (rice); Klein etal. (1988) Proc. Natl. Acad. Sci. USA 85, 4305-4309 (maize); Klein etal. (1988) Biotechnology 6, 559-563 (maize); Tomes, U.S. Pat. No.5,240,855; Buising et al., U.S. Pat. Nos. 5,322,783 and 5,324,646; Kleinet al. (1988) Plant Physiol. 91, 440-444 (maize); Fromm et al. (1990)Biotechnology 8, 833-839 (maize); Hooykaas-Van Slogteren et al. (1984)Nature 311, 763-764; Bowen et al., U.S. Pat. No. 5,736,369 (cereals);Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA 84, 5345-5349(Liliaceae); De Wet et al. (1985) in The Experimental Manipulation ofOvule Tissues, ed. Chapman et al. (Longman, New York), pp. 197-209(pollen); Kaeppler et al. (1990) Plant Cell Reports 9, 415-418 andKaeppler et al. (1992) Theor. Appl. Genet. 84, 560-566 (whisker-mediatedtransformation); D'Halluin et al. (1992) Plant Cell 4, 1495-1505(electroporation); Li et al. (1993) Plant Cell Reports 12, 250-255 andChristou and Ford (1995) Annals of Botany 75, 407-413 (rice); Osjoda etal. (1996) Nature Biotechnology 14, 745-750 (maize via Agrobacteriumtumefaciens); U.S. Pat. No. 5,736,369 (meristem transformation); andU.S. Pat. Nos. 5,302,523 and 5,464,765 (whiskers technology).

Nucleic acids of the invention may be introduced into plants bycontacting plants with a virus or viral nucleic acids. Generally, suchmethods involve incorporating an expression construct of the inventionwithin a viral DNA or RNA molecule. Further, it is recognized thatuseful promoters encompass promoters utilized for transcription by viralRNA polymerases. Methods for introducing expression constructs intoplants and expressing a protein encoded therein, involving viral DNA orRNA molecules, are known in the art. See, e.g., U.S. Pat. Nos.5,889,191; 5,889,190; 5,866,785; 5,589,367; and 5,316,931.

DNA constructs containing siRNAs may be integrated of the into the hostcell genome according to conventional methods, e.g., by homologousrecombination or other methods of integration, including targetedintegration at a particular host chromosomal site.

In other aspects of the invention, transient expression may be desired.In those cases, standard transient transformation techniques may beused, such as viral transformation methods, and microinjection of DNA orRNA, as well other methods well known in the art.

The cells from the plants that have stably incorporated the nucleotidesequence may be grown into plants in accordance with conventionaltechniques. See, e.g., McCormick et al. (1986) Plant Cell Reports 5,81-84. These plants may then be grown, and either pollinated with thesame transformed strain or different strains, and the resulting hybridhaving constitutive expression of the desired phenotypic characteristicimparted by the nucleotide sequence of interest and/or the geneticmarkers contained within the target site or transfer cassette. Two ormore generations may be grown to ensure that expression of the desiredphenotypic characteristic is stably maintained and inherited, and thenseeds are harvested to ensure expression of the desired phenotypiccharacteristic has been achieved.

Initial identification and selection of cells and/or plants comprisingsiRNA expression constructs may be facilitated by the use of markergenes. Gene targeting can be performed without selection if there is asensitive method for identifying recombinants, for example if thetargeted gene modification can be easily detected by PCR analysis, or ifit results in a certain phenotype. However, in most cases,identification of gene targeting events will be facilitated by the useof markers. Useful markers include positive and negative selectablemarkers as well as markers that facilitate screening, such as visualmarkers. Selectable markers include genes carrying resistance to anantibiotic such as spectinomycin, (e.g., the aada gene, Svab et al.(1990) Plant Mol. Biol. 14, 197); streptomycin, (Jones et al. (1987)Mol. Gen. Genet. 210, 86); kanamycin (e.g., nptII, Fraley et al. (1983)Proc. Natl. Acad. Sci. USA 80, 4803); hygromycin (e.g., HPT, VandenElzen et al. (1985) Plant Mol. Biol. 5, 299); gentamycin (Hayford et al.(1988) Plant Physiol. 86, 1216); phleomycin, zeocin, or bleomycin (Hilleet al. (1986) Plant Mol. Biol. 7, 171); or resistance to a herbicidesuch as phosphinothricin (bar gene); or sulfonylurea (acetolactatesynthase (ALS)) (Charest et al. (1990) Plant Cell Rep. 8, 643); genesthat fulfill a growth requirement on an incomplete media such as HIS3,LEU2, URA3, LYS2, and TRP1 genes in yeast; and other such genes known inthe art. Negative selectable markers include cytosine deaminase (codA)(Stougaard (1993) Plant J. 3, 755-761); tms2 (DePicker et al. (1988)Plant Cell Rep. 7, 63-66); nitrate reductase (Nussame et al. (1991)Plant J. 1, 267-274), SU1 (O'Keefe et al. (1994) Plant Physiol. 105,473-482); aux-2 from the Ti plasmid of Agrobacterium; and thymidinekinase. Screenable markers include fluorescent proteins such as greenfluorescent protein (GFP) (Chalfie et al. (1994) Science 263, 802; U.S.Pat. No. 6,146,826; U.S. Pat. No. 5,491,084; and PCT InternationalPublication No. WO 97/41228); reporter enzymes such as 13-glucuronidase(GUS) (Jefferson R. A. (1987) Plant Mol. Biol. Rep. 5, 387, U.S. Pat.No. 5,599,670, and U.S. Pat. No. 5,432,081), 13-galactosidase (lacZ);alkaline phosphatase (AP); glutathione S-transferase (GST) andluciferase (U.S. Pat. No. 5,674,713; and Ow et al. (1986) Science 234:856-859), visual markers like anthocyanins such as CRC (Ludwig et al.(1990) Science 247: 449-450) R gene family (e.g., Lc, P, S); A, C, R-nj,body and/or eye color genes in Drosophila, coat color genes in mammaliansystems, and others known in the art.

One or more markers may be used in order to select and screen fortargeting of a siRNA to a particular genomic locus, which is alsoreferred to as site-specific integration. One common strategy forsite-specific integration involves using a promoterless selectablemarker. Since the selectable marker lacks a promoter, random integrationevents generally do not lead to transcription of the gene. Genetargeting events will put the selectable marker under control of apromoter at the target site. Gene targeting events are identified byselection for expression of the selectable marker. Another commonstrategy utilizes a positive-negative selection scheme. This schemeutilizes two selectable markers, one that confers resistance (R⁺)coupled with one that confers sensitivity (S⁺), each with a promoter.When a heterologous nucleic acid containing the two markers is randomlyinserted, the resulting phenotype is R⁺/S⁺. When a gene-targeting eventis generated, the two markers are uncoupled and the resulting phenotypeis R⁺/S⁻. Examples of using positive-negative selection are found inThykjer et al. (1997) Plant Mol. Biol. 35, 523-530; and PCTInternational Publication No. WO 01/66717.

While various transformation methods are taught herein as separatemethods, the skilled artisan will readily recognize that certain methodscan be used in combination to enhance the efficiency of thetransformation process. Non-limiting examples of such methods includebombardment with Agrobacterium-coated microparticles (EP486234) ormicroprojectile bombardment to induce wounding followed byco-cultivation with Agrobacterium (EP486233).

Direct delivery can also be used to transform hosts according to theinvention disclosed herein. By way of non-limiting example, such directdelivery methods include polyethylene glycol treatment, electroporation,liposome mediated DNA uptake or the vortexing method. See, e.g., Freemanet al. (1984) Plant Cell Physiol. 29, 1353 and Kindle, (1990) Proc.Natl. Acad. Sci. USA 87, 1228. One form of direct DNA delivery is directgene transfer into protoplasts from embryogenic cell suspensioncultures. See Lazzeri and Lorz (1988) Advances in Cell Culture, Vol. 6,Academic Press, p. 291; OziasAkins and Lorz (1984) Trends inBiotechnology 2, 119.

The skilled artisan is aware of certain challenges of genotype-dependenttransformation arising from low regeneration potential of cereals.Accordingly, in one embodiment of the invention, transformation isaccomplished by a genotype-independent transformation approach based onthe pollination pathway. Ohta (1986) Proc. Natl. Acad. Sci. USA 83,715-719. In maize, high efficiency genetic transformation can beachieved by a mixture of pollen and exogenous DNA. Luo and Wu (1989)Plant Mol. Biol. Rep. 7, 69-77. Maize can be bred by bothself-pollination and cross-pollination techniques. Maize has separatemale and female flowers on the same plant, located on the tassel and theear, respectively. Natural pollination occurs in maize when wind blowspollen from the tassels to the silks that protrude from the tops of theears.

Transformation of tomato and melon with heterologous polynucleotidesaccording to the invention can be accomplished into intact plants viapollination pathway. See Chesnokov, et al. (1999) USSR Patent No.1708849; Bulletin of the USSR Patents, No. 4; Chesnokov and Korol(1993); Genetika USSR, 29, 1345-1355. The procedures of genetictransformation based on the pollination-fecundation pathway include: (i)employment of a mixture (paste) of the pollen and transforming DNA; (ii)delivery of the alien DNA into the pollen tube, after pollination; and(iii) microparticle bombardment of microspores or pollen grains.

In one aspect of the invention, plants hosts are transformed usingAgrobacterium technology (e.g., A. tumefaciens and A. rhizogenes).Agrobacterium-mediated transfer is a widely applicable system forintroducing genes into plant cells because the DNA can be introducedinto whole plant tissues, thereby bypassing the need for regeneration ofan intact plant from a protoplast. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art. See, e.g., the methods described by Lloyd et al.(1986) Science 234, 464-466; Horsch et al. (1987)“Agrobacterium-mediated transformation of plants,” Plant Biology Alan R.Liss, NY pp 317-329; and Wang (2006) Agrobacterium protocols, Vol. 2,Humana Press, Totowa N.J. and U.S. Pat. No. 5,563,055.

Agrobacterium-mediated transformation can efficiently be used withdicotyledonous host plants of the invention including, by way ofnon-limiting example, Arabidopsis, corn, soybean, cotton, canola,tobacco, tomato, and potato.

Agrobacterium-mediated transformation is also applicable to nearly allmonocotyledonous plants of the invention. By non-limiting example, suchmonocotyledonous plant technologies are adaptable to rice, wheat, andbarley. See, e.g., Hiei et al. (1994) Plant J. 6, 271-282; Zhang et al.(1997) Mol. Biotechnol. 8, 223-231; Ishida et al. (1996) Nat.Biotechnol. 14, 745-750; McCormac et al. (1998) Euphytica 99, 17-25,Tingay S. et al. (1997) Plant J. 11, 1369-1376; and U.S. Pat. No.5,591,616.

Agrobacterium-mediated transformation can be accomplished with culturedisolated protoplasts or by transformation of intact cells or tissues.Agrobacterium-mediated transformation in dicotyledons facilitates thedelivery of larger pieces of heterologous nucleic acid as compared withother transformation methods such as particle bombardment,electroporation, polyethylene glycol-mediated transformation methods,and the like. In addition, Agrobacterium-mediated transformation appearsto result in relatively few gene rearrangements and more typicallyresults in the integration of low numbers of gene copies into the plantchromosome.

Modern Agrobacterium transformation vectors are capable of replicationin E. coli as well as Agrobacterium, allowing for convenientmanipulations as described. Klee et al. (1987) Ann. Rev. PlantPhysiology 38, 467-486. Moreover, recent technological advances invectors for Agrobacterium-mediated gene transfer have improved thearrangement of genes and restriction sites in the vectors to facilitatethe construction of vectors capable of expressing variouspolypeptide-coding genes. The vectors described by Horsch et al. haveconvenient multi-linker regions flanked by a promoter and apolyadenylation site for direct expression of inserted polypeptidecoding genes and are suitable for present purposes. Horsch et al. (1987)“Agrobacterium-mediated transformation of plants,” Plant Biology Alan R.Liss, NY pp 317-329. In addition, Agrobacterium containing both armedand disarmed Ti genes can be used for the transformations. In thoseplant strains where Agrobacterium-mediated transformation is efficient,it is the method of choice because of the facile and defined nature ofthe gene transfer.

When Agrobacteria are used to transform plant cells according to theinvention, nucleic acids to be inserted can be cloned into specialplasmids, namely either into an intermediate vector or into a binaryvector. The intermediate vectors can be integrated into the Ti or Riplasmid by homologous recombination owing to sequences that arehomologous to sequences in the T-DNA. The Ti or Ri plasmid alsocomprises the vir region necessary for the transfer of the T-DNA.Intermediate vectors cannot replicate themselves in Agrobacteria. Theintermediate vector can be transferred into Agrobacterium tumefaciens bymeans of a helper plasmid (conjugation).

Binary vectors can replicate themselves both in E. coli and inAgrobacteria. Such vectors can comprise a selection marker gene and alinker or polylinker, which are framed by the right and left T-DNAborder regions. They can be transformed directly into Agrobacteria.Holsters et al. (1978) Mol. Gen. Genet. 163, 181-187. The Agrobacteriumused as host cell can comprise a plasmid carrying a vir region. The virregion is necessary for the transfer of the T-DNA into the plant cell.Additional T-DNA may be contained. The bacterium so transformed is usedfor the transformation of plant cells. Plant explants can advantageouslybe cultivated with Agrobacterium tumefaciens or Agrobacterium rhizogenesfor the transfer of the DNA into the plant cell. Whole plants can thenbe regenerated from the infected plant material (for example, pieces ofleaf, segments of stalk, roots, but also protoplasts orsuspension-cultivated cells) in a suitable medium, which may containantibiotics or biocides for selection. The plants so obtained can thenbe tested for the presence of the inserted nucleic acids.

Methods of Conferring Desirable Traits

The invention further provides methods of identifying a siRNA thatconfers a desirable phenotypic outcome in a target organism. In oneaspect of the invention, the method comprises (a) contacting a targetorganism with a siRNA molecule of a siRNA library as described herein;and (b) correlating the siRNA treatment of (a) with a desirablephenotypic outcome. For example, siRNAs that confer resistance tosoybean cyst nematode were identified by (a) contacting soybean cystnematode with a siRNA molecule of a siRNA library of the invention; and(b) correlating the siRNA treatment of (a) with soybean resistance tosoybean cyst nematode infection. See Examples 2 and 4.

The phrase “correlating the siRNA treatment” as used herein, refers tothe process of measuring the effects of contacting a target organismwith a siRNA molecule and determining whether a desirable phenotypicoutcome has been achieved in the target organism by means of such siRNAtreatment. In general, correlation of a siRNA treatment is measuredrelative to a control treatment.

For example, transgenic soybean hairy roots expressing siRNAs werecontacted with soybean cyst nematodes (SCN). The number of SCN cystsformed in multiple independent, biologically replicated experiments weredetermined and compared to controls that did not express siRNAs.Statistically significant reductions in the number of cysts formed wereobserved during the experiments compared with controls. Consequently,the siRNA expression correlated with reduced infectivity of SCN, i.e.,soybean resistance to SCN infection. See Examples 2 and 4.

As used herein, the terms “contacting” and “administering,” or phrase“contact with” are used interchangeably, and refer to a process by whichthe siRNAs or miRNAs of the invention are delivered or administered totarget organisms, in order to inhibit expression of a gene in the targetorganisms. Contacting describes physical proximity of siRNAs or miRNAsand the target organism so that they interact. The siRNAs or miRNAs maybe administered or delivered in any number of ways, including, but notlimited to, direct introduction into a cell (i.e., intracellularly); orextracellular introduction into a cavity, interstitial space, or intothe circulation of the target organism, oral introduction, the siRNA ormiRNA may be introduced by bathing the target organism in a solutioncontaining siRNA or miRNA, or the siRNA or miRNA may be present in afood source. Methods for oral introduction include direct mixing ofsiRNA or miRNA with a food source of the target organism, as well asengineered approaches in which a species that is used as food isengineered to express a siRNA or miRNA, and then this species is fed tothe target organism to be affected. For example, the siRNA or miRNAconstructs may be sprayed onto a plant, or the siRNA may be applied tosoil in the vicinity of roots, taken up by plant and/or the targetorganism, or a plant may be genetically engineered to express the siRNAor miRNA in an amount sufficient to kill or adversely affect some or allof the target organisms to which the plant is exposed. Thus,“contacting” refers to any process by which a siRNA or miRNA isadministered or delivered to a target organism to thereby inhibitexpression of a gene in the target organism.

As used herein, “contacting” also refers to placing a pest, pathogen, ortarget organism on or near a host plant, or part thereof, such that thepest, pathogen, or target organism has an opportunity to interact with,attack, or infect the plant or plant part, which effectively results inproximity between siRNAs expressed in the host plant and the targetorganism.

The siRNA may be “contacted” or “administered” to the target in anymanner that results in physical proximity of a siRNA and a targetnucleic acid permitting interaction. In one aspect of the invention, asiRNA may be expressed within a host organism and then passively diffuseor be actively transported to a target organism. Expression within thehost can be transient, or stable, and/or inducible. The siRNA can beexpressed as a precursor or inactive form that becomes active within thetarget organism. Expression in a host may be achieved using any of theexpression constructs and vectors described herein.

Other examples of contacting include, but are not limited to, directintroduction into a cell (i.e., intracellularly); extracellularintroduction into a cavity, interstitial space, or into the circulationof a target organism; oral introduction; the siRNA may be introduced bybathing or soaking the target organism in a solution containing siRNA.Methods for oral introduction include direct mixing of siRNA with foodof a target organism, as well as engineered approaches in which aspecies that is used as food is engineered to express a siRNA, and thenfed to the organism to be affected.

Where the target organism or host organism is a plant, a compositioncomprising a siRNA may be sprayed onto the plant, or the siRNA may beapplied to soil in the vicinity of roots, taken up by the plant and/ortarget pest or pathogen, or a plant may be modified to express thesiRNA.

A host organism expressing a heterologous siRNA is “transgenic.” As usedherein, the term “transgenic” refers to a host organism, or part or cellthereof, which comprises within its genome a heterologouspolynucleotide. A transgenic host organism may be stably transformed ortransiently transformed. If the heterologous siRNA is stably integratedwithin the genome, it is passed on, or heritable, to successivegenerations. The heterologous siRNA may be integrated into the genomealone or as part of an expression construct. Transgenic is used hereinto include any cell, cell line, callus, tissue, plant part or plant, thegenotype of which has been altered by the presence of a heterologousnucleic acid including those transgenics initially so altered as well asthose created by breeding, sexual crosses, or asexual propagation fromthe initial transgenic cell.

The phrase “desirable phenotype,” as used herein, refers to an intendedeffect that has been elicited in a target organism and/or host organismas a result of siRNA gene silencing or suppression. The inventionprovides methods for identifying siRNAs that confer desirablephenotypes. In one aspect of the invention, the method comprisesdesigning siRNAs complementary to target genes whose regulation is knownto have a desirable effect. For example, where the target organism is aplant pest or pathogen, the method can comprise designing siRNAscomplementary to target genes involved in the development, survival, orpathogenicity of the plant pest or pathogen. In another aspect of theinvention, the method comprises empirically identifying siRNAs capableof eliciting a desired phenotype by screening siRNA libraries asdescribed herein. See Examples 2 and 4. In addition, these two generalapproaches may be combined.

Where the host or target organism is a plant, desirable phenotypesinclude resistance to a pest or pathogen, resistance to abiotic stress,and improved growth or yield. Where the target organism is a plant pestor pathogen, desirable phenotypes include reduced infectivity, decreasedpersistence, reduced disease causing ability, or death of the pest.

“Resistance to a target organism,” as used herein, refers to the abilityof a host organism to withstand or reduce the severity of pest orpathogen distress, infections, or disease. Resistance can be measured bythe host's ability to survive pest infection, reduced pestsusceptibility, reduced pest burden, increased yields, decreasedattrition or death, or other suitable agronomic indicators.

As used herein, the phrases “abiotic stress,” “stress,” or “stresscondition” refer to the exposure of a plant, plant part, plant cell, orthe like, to a non-living, i.e., abiotic physical stress, chemicalagents, or environmental conditions that can produce adverse effects onmetabolism, growth, development, propagation, and/or survival of theplant (collectively “growth”). Abiotic stress can be imposed on a plant,for example, because of environmental factors such as water (e.g.,flooding, drought, and dehydration), anaerobic conditions (e.g., a lowlevel of oxygen), abnormal osmotic conditions, salinity or temperature(e.g., hot/heat, cold, freezing, frost), a deficiency of nutrients,exposure to pollutants, or by a exposure to hormone, second messenger orother molecule. Anaerobic stress, for example, is due to a reduction inoxygen levels (hypoxia or anoxia) sufficient to produce a stressresponse. A flooding stress can be due to prolonged or transientimmersion of a plant, plant part, tissue, or isolated cell in a liquidmedium such as occurs during a monsoon, wet season, flash flooding, orexcessive irrigation of plants, or the like. A cold stress or heatstress can occur due to a decrease or increase, respectively, in thetemperature from the optimum range of growth temperatures for aparticular plant species. Such optimum growth temperature ranges arereadily determined or known to those skilled in the art. Dehydrationstress can be induced by the loss of water, reduced turgor, or reducedwater content of a cell, tissue, organ, plant part, or whole plant.Drought stress can be induced by or associated with the deprivation ofwater or reduced supply of water to a cell, tissue, organ, or organism.Salinity-induced stress (i.e., salt-stress) can be associated with orinduced by a perturbation in the osmotic potential of the intracellularor extracellular environment of a cell.

As used herein, “resistance to abiotic stress,” “abiotic stressresistance,” or “abiotic stress tolerance” includes, but is not limitedto, greater water optimization; greater tolerance to dehydration, waterdeficit conditions, or drought; better recovery from dehydration, waterdeficit conditions, or drought; increased root growth; increased lateralroot formation; increased root branching; increased surface area ofroots; increased root mass; more root hairs; increased nutrient uptake;increased micronutrient uptake; increased metabolic efficiency; greaterphotosynthetic capacity; more rapid growth rate; greater fruit or seedyield; modified plant architecture; enhanced herbicide resistance;reduced or increased height; reduced or increased branching; enhancedcold and frost tolerance; improved vigor; enhanced color; enhancedhealth and nutritional characteristics; improved storage; enhancedyield; enhanced salt tolerance; enhanced resistance of wood or planttissue to decay; enhanced heavy metal tolerance; enhanced sweetness;improved texture; decreased phosphate content; increased germination;improved starch composition; improved flower longevity; production ofnovel resins; production of novel proteins or peptides; enhancedagronomic traits, or any other agronomically desirable or commerciallyadvantageous traits or characteristics.

The skilled artisan can readily identify pest or pathogen genes totarget using the invention disclosed herein. Such a target gene could beany pest gene that serves a direct or indirect role in such a pest'sdeleterious effects on a host plant. By way of example only, such a genemay be one that serves a role in pest growth, development, replicationand reproduction, and invasion or infection.

Target genes for use in the invention may include, for example, thosethat play important roles in the viability, growth, development,reproduction and infectivity of a particular pest. These target genesmay be one or more of any housekeeping genes, transcription factors, orpest- or pathogen-specific genes that provide an observable phenotype,in particular a phenotype that results in the suppression of response tostimuli, movement, feeding, growth, development, reproduction, andinfectivity or eventually results in the death of the pest or pathogen.

The genes targeted for suppression can also include those required foressential functions such as DNA replication, RNA transcription, proteinsynthesis, amino acid biosynthesis, amino acid degradation, nucleotidesynthesis, nucleotide degradation, muscle formation, juvenile hormoneformation, juvenile hormone regulation, ion regulation and transport,digestive enzyme synthesis, maintenance of cell membrane potential,sperm formation, pheromone synthesis, pheromone sensing, antennaeformation, wing formation, leg formation, egg formation, larvalmaturation, digestive enzyme formation, haemolymph synthesis, haemolymphmaintenance, neurotransmission, cell division, energy metabolism,development and differentiation, respiration, and apoptosis.

For example, target genes that are presumed to be effective in producingsuch phenotypes are similar to those that have been shown to affect theviability, growth, development, mobility, neurological stimulation,muscular function, and reproduction in C. elegans, including but notlimited to the following phenotypes: (Adl) adult lethal, (Age), (Bli)blistered, (Bmd) body morphology defect, (Ced) Cell death abnormality,(Clr) clear, (Dan DAuer Formation, (Dpy) dumpy, (Egl) egg laying defect,(Emb) embryonic lethal, (Evl) everted vulva, (Fern) feminization of XXand XO animals, (Fgc) Fewer Germ Cells, (Fog) feminization of germline,(Gon) GONad development abnormal, (Gro) slow growth, (Him) highincidence of male progeny, (Hya) HYperActive, (Let) larval lethal, (Lin)lineage abnormal, (Lon) long body, (Lpd), (Lva) larval arrest, (Lvl)larval lethal, (Mab) Male ABnormal, (Mei) Defective meiosis, (Mig)MIGration of cells abnormal, (Mlt) molt defect, (Morphology), (Mut)Mutator, (Muv) MUlti-Vulva, (Oma) Oocyte MAturation defective, (Pat)Paralyzed, Arrested elongation at Two-fold, (Pch) PatCHy coloration,(Pnm) Pronuclear migration alteration in early embryo, (Prl) paralyzed,(Prz) PaRaLyzed, (Pvl) protruding vulva, (Pvu) protruding vulva, (Rde),(Reproductive), (Rol) roller, (Rot) centrosome pair and associatedpronuclear rotation abnormal, (Rup) exploded, (Sck) sick, (Sle) Slowembryonic development, (Slu) SLUggish, (Sma) small, (Spd) SpinDle,abnormal embryonic, (Spo) Abnormal embryonic spindle position andorientation, (Step) sterile, (Stp) sterile progeny, (Unc) uncoordinated,(Unclassified), (Vul) vulvaless, (WT), (defect) morphological orbehavioral defects.

As further examples, potential Coleoptera target genes include:swelling-dependent chloride channels; glyceraldehyde-3-phosphatedehydrogenase; glucose-6-phosphate 1-dehydrogenase; chitinase; vacuolarATPase D subunit 1; ADP-ribosylation factor; juvenile hormone esterase;transcription factor IIB; cytosolic juvenile hormone binding protein;actin orthologs; chitinase; α-tubulin; vacuolar ATPase A subunit 2;vacuolar ATPase E; ATP synthase chain A; endoglucanase; ADP/ATPtranslocase; activating transcription factor; mRNA capping enzyme; appleATPase2; ribosomal protein L9; ribosomal protein L19; 26S proteosomeregulatory subunit p28; chromodomain helicase-DNA-binding protein; andβ-tubulin. See U.S. Pat. No. 7,812,219 and Baum et al. (2007) Nat.Biotech. 25, 1322-1326, including Table 1 in the supplementaryinformation, all of which are incorporated by reference herein in theirentirety.

Additional target genes encode various gene products that, whendisrupted, exert a negative effect or observable phenotype in Drosophilaor in C. elegans.

Further target genes in various organisms are listed in Table 6.

TABLE 6 Target Genes Pest or Pathogen Pest Pathogenecity Gene FungiCutinases Kinases Ribosomal RNAs Adhesins Elicitins Bacteria CutinasesMacerating enzymes Kinases Ribosomal RNAs Adhesins G-proteins InsectsKinases Ribosomal RNAs G-proteins Moulting factors Serine proteasesCysteine proteases Juvenile hormone esterase Nematodes Kinases RibosomalRNAs G-proteins Cuticle collagen proteins Cathepsin proteases VirusesCapsid, coat proteins Viral polymerases Viral nucleic acid bindingproteins Viral packaging proteins Viral proteases Viral proteins,generally Viral genomic nucleic acids

The invention further provides a siRNA molecule that targets both anematode gene, such as a soybean cyst nematode gene, and an endogenousplant gene related to a nematode-resistant plant phenotype. In oneembodiment the siRNA molecule is capable of suppressing expression ofthe nematode gene and the endogenous plant gene. In another embodiment,the siRNA when expressed in a transgenic plant, or part thereof, confersupon the plant, or part thereof, a level of tolerance to nematodeinfection that is greater than would be expected from suppression of thenematode gene or the endogenous plant gene alone. In yet anotherembodiment, the endogenous plant gene is an ethylene response gene. Inparticular, siRNAs designed to target nematode genes that are alsocapable of modulating gene silencing of ethylene response (ETR) nucleicacids, such as, ETR1, EIN1, QITR, Q8, TETR, TGETR1, TGETR2 and the likeare encompassed by the invention.

A number of ethylene response genes have been characterized. The ETR1gene from Arabidopsis, as well as other plant homologues of ETR1 andETR2, are considered to be ethylene receptors. (see, e.g., Gamble et al.(1998) PNAS USA 95, 7825-7829). The Arabidospsis ETR1 protein containsan amino-terminal half with a hydrophobic domain responsible forethylene binding and membrane localization (Gamble et al. supra). Thecarboxyl-terminal half of the Arabidopsis ETR1 contains domains withhomology to histidine kinases and response regulators (Gamble et al.,supra).

Ethylene production in plants is involved in a plant's response tomultiple biotic and abiotic stresses. Plants carrying mutations in ETRgenes have been studied. For example, ethylene insensitive soybeanplants with mutations in the ETR1 gene have been found to have increasedresistance to some pathogens but reduced resistance to other pathogens(Hoffman et al., (1999) Plant Physiology 119, 935-949). In addition,alteration in ethylene sensistivity in soybean has been implicated intolerance to soybean cyst nematode (Bent et al. 2006. Crop Science46:893-901).

In another embodiment, the ETR1 gene of the invention is a soybean ETR1.In another embodiment, the soybean ETR1 gene comprises SEQ ID NO: 52, ora complement thereof.

In another embodiment, the siRNA molecule that targets a soybean cystnematode gene and a soybean ETR1 gene comprises SEQ ID NO: 3 (siRNA0097)or SEQ ID NO: 4 (siRNA00145). In yet another embodiment the star strandof the siRNA targets ETR1 and comprises SEQ ID NO: 55 (siRNA0097*) orSEQ ID NO: 56 (siRNA0145*). In yet another embodiment, the mRNA portionof the soybean ETR1 gene that binds to siRNA0097 (SEQ ID NO: 3) andsiRNA0145 (SEQ ID NO: 4) comprises SEQ ID NO: 53. In still anotherembodiment, the mRNA portion of the soybean ETR1 gene that bindssiRNA0097* (SEQ ID NO: 55) and siRNA0145* (SEQ ID NO: 56) comprises SEQID NO: 54.

In another embodiment, the invention encompasses an siRNA moleculedesigned to target a gene of a nematode plant pest that when contactedwith the nematode pest the nematode pest has decreased capability toinfect a plant susceptibile to infection by the nematode, and whereinthe siRNA, when expressed in the plant, suppresses expression of anendogenous plant gene, wherein the suppression of the plant gene confersupon the plant resistance to the nematode plant pest. In anotherembodiment, the endogenous plant gene is an ethylene response gene. Inyet another embodiment, the ethylene response gene is a soybean ETR1gene. In another embodiment, the soybean ETR1 gene comprises SEQ ID NO:52. In another embodiment, the pest nematode is soybean cyst nematode.In still another embodiment, the siRNA is selected from the groupconsisting of SEQ ID NO: 3 (siRNA0097), SEQ ID NO: 4 (siRNA0145), SEQ IDNO: 55 (siRNA0097*) and SEQ ID NO: 56 (siRNA0145*). In still anotherembodiment, the level of expression of the ETR1 gene in the transgenicplant is suppressed by at least about 30% compared to a wild-type plantof the same species.

In another embodiment, the invention encompasses a siRNA moleculedesigned to target a gene of a nematode plant pest, wherein the siRNAmolecule is capable of suppressing the expression of the nematode geneand an endogenous plant gene, whereby the suppression of the nematodegene and the endogenous plant gene confer upon a transgenic plant orpart thereof expressing the siRNA molecule resistance to the nematode.In another embodiment, the endogenous plant gene is an ethylene responsegene. In still another embodiment, the transgenic plant or part thereofis a soybean plant or part thereof. In yet another embodiment, theethylene response gene is a soybean ETR1 gene. In another embodiment,the soybean ETR1 gene comprises SEQ ID NO: 52. In another embodiment,the nematode is soybean cyst nematode. In still another embodiment, thesiRNA is selected from the group consisting of SEQ ID NO: 3 (siRNA0097),SEQ ID NO: 4 (siRNA0145), SEQ ID NO: 55 (siRNA0097*) and SEQ ID NO: 56(siRNA0145*). In still another embodiment, the level of expression ofthe ETR1 gene in the transgenic plant or part thereof is suppressed byat least about 30% compared to a non-transgenic plant or part thereof ofthe same species.

In one embodiment, the invention provides a transgenic plant, or partthereof, having a reduced level of expression of a ethylene responsegene compared to a non-transgenic plant, or part thereof, of the samespecies, wherein the transgenic plant or part thereof comprises an siRNAthat suppresses the expression of a nematode pest gene, and wherein thetransgenic plant has a greater tolerance to infection by the nematodepest than would be expected from the reduced level of expression of theethylene response gene or the suppression of the nematode gene alone. Inanother embodiment, the transgenic plant is a soybean plant. In anotherembodiment, the ethylene response gene is a soybean ETR1 gene. Inanother embodiment, the ETR1 gene comprises SEQ ID NO: 52. In anotherembodiment, the pest nematode is soybean cyst nematode. In still anotherembodiment, the siRNA is selected from the group consisting of SEQ IDNO: 3 (siRNA0097), SEQ ID NO: 4 (siRNA0145), SEQ ID NO: 55 (siRNA0097*)and SEQ ID NO: 56 (siRNA0145*). In still another embodiment, the levelof expression of the ETR1 gene in the transgenic plant is reduced by atleast about 30%. In yet another embodiment, the greater tolerance toinfection by the soybean cyst nematode is measured by the number ofcysts on soybean roots. In another embodiment, the number of cysts onthe roots is reduced by at least about 52%.

In one embodiment, the invention encompasses a method of conferringnematode pest resistance to a plant, or part thereof, comprisingexpressing in the plant or part thereof a nucleic acid moleculecomprising an siRNA that suppresses the expression of a nematode pestgene, and wherein the plant or part thereof is ethylene-insensitive,whereby the plant or part thereof is resistant to the nematode pest to agreater degree than would be expected from the siRNA or ethyleneinsensitivity alone. In another embodiment, the plant or part thereof isa soybean plant. In another embodiment, the ethylene-insensitivity isdue to the suppression of an ETR1 gene (ethylene response gene). Inanother embodiment, the ETR1 gene comprises SEQ ID NO: 52. In anotherembodiment, the nematode pest is soybean cyst nematode. In still anotherembodiment, the siRNA is selected from the group consisting of SEQ IDNO: 3 (siRNA0097), SEQ ID NO: 4 (siRNA0145), SEQ ID NO: 55 (siRNA0097*)and SEQ ID NO: 56 (siRNA0145*). In still another embodiment, the levelof expression of the ETR1 gene in the plant or part thereof is reducedby at least about 30%. In yet another embodiment, the resistance toinfection by the soybean cyst nematode is measured by the number ofcysts on soybean roots. In another embodiment, the number of cysts onthe roots is reduced by at least about 52%.

In anther embodiment, the invention encompasses a method of enhancingresistance of a plant, or part thereof, to infection by a nematode pest,comprising introducing into the plant, or part thereof, a nucleic acidcomprising a siRNA that suppresses the expression of a nematode genethereby reducing the ability of the nematode to infect the plant, orpart thereof, wherein the plant, or part thereof, additionally has areduced level of expression of an ethylene response gene compared to aplant, or part thereof, of the same species without the siRNA, wherebythe plant or part thereof comprising the siRNA has a greater resistanceto infection by the nematode than would be expected from the suppressionof the nematode gene or the suppression of the ethylene response genealone. In another embodiment, the plant or part thereof is a soybeanplant. In another embodiment, the ethylene response gene is an ETR1gene. In another embodiment, the ETR1 gene comprises SEQ ID NO: 52. Inanother embodiment, the nematode pest is soybean cyst nematode. In stillanother embodiment, the siRNA is selected from the group consisting ofSEQ ID NO: 3 (siRNA0097), SEQ ID NO: 4 (siRNA0145), SEQ ID NO: 55(siRNA0097*) and SEQ ID NO: 56 (siRNA0145*). In still anotherembodiment, the level of expression of the ETR1 gene in the plant orpart thereof is reduced by at least about 30%. In yet anotherembodiment, the greater resistance to infection by the soybean cystnematode is measured by the number of cysts on soybean roots. In anotherembodiment, the number of cysts on the roots is reduced by at leastabout 52%.

In still another embodiment, the invention encompasses a method ofreducing cyst development on soybean roots susceptible to soybean cystnematode infection, comprising introducing into cells of a soybean plantor part thereof a nucleic acid molecule comprising a siRNA that whencontacted with the soybean cyst nematode causes the soybean cystnematode to produce a reduced number of cysts on the soybean roots andwherein the soybean plant or part thereof has a reduced level of an ETR1gene, whereby cyst development on soybean roots is reduced to a greaterdegree than would be expected from the siRNA contacting the soybean cystnematode or the reduced expression level of the ETR1 gene alone. Inanother embodiment, the ETR1 gene comprises SEQ ID NO: 52. In stillanother embodiment, the siRNA is selected from the group consisting ofSEQ ID NO: 3 (siRNA0097), SEQ ID NO: 4 (siRNA0145), SEQ ID NO: 55(siRNA0097*) and SEQ ID NO: 56 (siRNA0145*). In still anotherembodiment, the level of expression of the ETR1 gene in the plant orpart thereof is reduced by at least about 30%. In another embodiment,the number of cysts on the roots is reduced by at least about 52%.

EXAMPLES

The foregoing description of the aspects, including preferred aspects,of the invention has been presented only for the purpose of illustrationand description and is not intended to be exhaustive or to limit theinvention to the precise forms disclosed. Numerous modifications andadaptations thereof will be apparent to those skilled in the art withoutdeparting from the spirit and scope of the invention.

Example 1 Soybean Cyst Nematode siRNA Library Design and Construction

A small interfering RNA library was prepared having a partiallyrandomized seed sequence to target mRNAs of a pest or pathogen. Thesoybean cyst nematode (SCN) was chosen as a target pest for testing thissiRNA library.

A 21-nucleotide small interfering RNA library was designed with arandomized seed sequence located at positions 2-8 from the 5′-end, andpositions 1, 9-21 were fixed. Since the small RNA was designed to targetnematode genes, the non-seed sequence was based on Caenorhabditiselegans microRNAs. Bioinformatic analyses of the predicted and known C.elegans miRNAs revealed conserved nucleotides at each position of thenon-seed region of miRNAs (i.e., positions 1 and 11-19). Thesenucleotides were selected for the non-seed sequence for the siRNAlibrary. Uridine residues were chosen for positions 20 and 21 in orderto increase the stability of the molecule for in vitro screening. Themodel non-seed sequence generated from the consensus C. elegans miRNA is5′-UNNNNNNNUGUUGAUCUGGUU-3′, (SEQ ID NO: 47) where N indicates a randomnucleotide (i.e., either A, C, G, or U) in the seed sequence. A siRNAlibrary of this exemplary sequence consists of 4⁷ (i.e., 4×4×4×4×4×4×4)different RNA molecules, or 16,384 possible sequences.

In order to reduce the complexity of an RNA library (i.e., the number ofsequences contained in the library), a subset of sequences were excludedfrom the library. In particular, the complexity of the siRNA library wasreduced by computationally excluding nucleotides that occurred at aparticular position in C. elegans miRNA seed sequences at lowerfrequencies. In this example, the frequency threshold was chosen to be20%. Accordingly, any nucleotide that was determined to occur less than20% at a particular position in a C. elegans seed sequence usingbioinformatic analyses was excluded at that particular position.Nucleotides that occurred with a frequency of 20% or greater in C.elegans seed sequences were included in the library. Table 7 shows thenucleotides that are frequently observed at each position.

TABLE 7 Nucleotides present in greater than 20% of C. elegans miRNA seedsequences Position in miRNA seed sequence 2 3 4 5 6 7 8 Nucleotide 1 A AC A G A A Nucleotide 2 G G G G U C G Nucleotide 3 — U — U — G U Numberof Nucleotides 2 3 2 3 2 3 3 Motif Seed Sequence R D S D K V D

The reduced combination of nucleotides at the 7-positions within theseed sequence was equal to: 2×3×2×3×2×3×3, or 648 possible sequences,which was a 25-fold reduction in complexity.

In addition, RNA sequences were excluded from the library if theresulting small RNA sequence contained homonulceotide quadruplets, suchas AAAA. Further, small RNA sequences having a GC-content in positions1-9 (i.e., position 1 and the seed sequence) greater than the GC-contentof positions 11-19 were also excluded. After these two additionalparameters were considered, the number of siRNA sequences in the librarywas reduced to 563 sequences. The siRNA consensus motif is5′-URDSDKVDUGUUGAUCUGGUU-3′ (SEQ ID NO: 48).

The 563 siRNAs were synthesized as duplexes using standard automatedsynthesis. In order to enhance the stability, the 3′-residues may bestabilized against nucleolytic degradation, e.g., they consist of purinenucleotides. Alternatively, substitution of pyrimidine nucleotides bymodified analogues, e.g., substitution of uridine by 2′-deoxythymidineis tolerated and does not affect the efficiency of RNA interference.siRNAs were synthesized with a dTdT dinucleotide at the 3′-end as anoverhang to increase stability and prevent nucleolytic degradation.

Example 2 In vitro Screening

Second stage juveniles (J2s) of soybean cyst nematodes weresurface-sterilized in 0.01% HgCl₂ and then rinsed in sterile water3-times before being resuspended in NGM medium (1.7% bacto-agar, 0.25%peptone, 0.3% NaCl, 1 mM MgSO₄, 1 mM CaCl₂, 25 mM KH₂PO₄, pH 6.0)containing 50 mM octopamine and a single siRNA duplex at a concentrationof 0.5 mg/mL (ca. 0.4 mM RNA duplex).

A 100 μL aliquot of the soaking solution containing 500 J2s wasdispensed into a single well on a 48-well plate, and incubated at 26° C.for 5 days. The J2s were observed daily. A siRNA duplex targeting the H.glycines hg-rps23 gene was used as a positive control. As a negativecontrol, soaking solution without siRNA duplex was used.

After 5 days of incubation, the J2s were observed for their activitycompared to the controls and then inoculated onto the roots of 4-day oldsoybean seedlings grown in germination pouches containing water-soakedpaper towels. The infected seedlings were cultured in a growth chamberat 26° C. with 16 hour per day lighting for a month.

The number of cysts formed on each plant were counted and compared tothe controls. The results indicated that 15 of the 563 duplexes testedreduced the number of SCN cysts on the root to less than 40% of thecontrols. The sequences of the siRNAs that reduced the SCN cysts arelisted in Table 8. The si-rps23-1 siRNA was used as a postitive controlbecause this RNA has been shown to reduce SCN cysts. See PCT ApplicationPCT/US11/064082.

TABLE 8 siRNA sequences reducing the incidence ofSCN cysts on soybean seedling roots siRNA ID siRNA Sequence (5′→3′)SEQ ID NO: siRNA0043 UAACUUAUUGUUGAUCUGGUU 1 siRNA0046UAACUUCUUGUUGAUCUGGUU 2 siRNA0097 UAAGUUCAUGUUGAUCUGGUU 3 siRNA0145UAGCUUGAUGUUGAUCUGGUU 4 siRNA0192 UAGGUUGGUGUUGAUCUGGUU 5 siRNA0243UAUCUUCGUGUUGAUCUGGUU 6 siRNA0309 UGACAGGAUGUUGAUCUGGUU 7 siRNA0382UGAGGUCAUGUUGAUCUGGUU 8 siRNA0423 UGGUAUGGUGUUGAUCUGGUU 9 siRNA0458UGGGAUCUUGUUGAUCUGGUU 10 siRNA0483 UGUCAGAUUGUUGAUCUGGUU 11 siRNA0514UGUCGUGAUGUUGAUCUGGUU 12 siRNA0531 UGUCUUCGUGUUGAUCUGGUU 13 siRNA0569UGUGUGAUUGUUGAUCUGGUU 14 si-rps23-1 UUCUCGGAAAUUGCGCUUCUU 15

Example 3 Construction of Target-Specific amiRNAs

The 15 siRNA molecules that reduced the number of SCN cysts on soybeanroots were assembled into artificial microRNA constructs. SoybeanmicroRNA precursor, gma-MIR164, was used as the backbone of the amiRNA.The miR164/miR164* sequence on this precursor was replaced bysiRNA/siRNA* sequence, while the mismatch positions on themiR164/miR164* duplex were maintained in the artificial siRNA/siRNA*sequence by making mutations on the siRNA* passenger strand.

The design of the artificial microRNA (amiRNA) for expression ofanti-SCN siRNA in host plant cell follows the literature of Schwab etal., where amiRNAs were designed to target individual genes or groups ofendogenous genes in a plant cell. See Schwab et al. (2006) Plant Cell18, 1121-1133; Alvarez et al. (2006) Plant Cell 18, 1134-1151. Thesoybean miRNA precursor gma-MIR164 was chosen for the backbone of theamiRNAs. Details regarding the assembly of the target-specificartificial microRNA are described in U.S. Provisional Application61/421,275 filed Dec. 9, 2010, which is incorporated by reference hereinin its entirety. The aMIR164-rps23-1 amiRNA was used as a positivecontrol as in the siRNA experiments.

TABLE 9 Target-specific amiRNA sequences siRNA IDamiRNA Sequence (5′→3′) SEQ ID NO: amiRNA0043AGCTCCTTGTTAACTTATTGTTGATCTGGCAAGTCTCTTGGATC 16TCAAATGCCACTGAACCCTTTGCCAGATCAAGTATAAGTTACAA CACGGGTTT amiRNA0046AGCTCCTTGTTAACTTCTTGTTGATCTGGCAAGTCTCTTGGATC 17TCAAATGCCACTGAACCCTTTGCCAGATCAAGTAGAAGTTACAA CACGGGTTT amiRNA0097AGCTCCTTGTTAAGTTCATGTTGATCTGGCAAGTCTCTTGGATC 18TCAAATGCCACTGAACCCTTTGCCAGATCAAGTTGAACTTACAA CACGGGTTT amiRNA0145AGCTCCTTGTTAGCTTGATGTTGATCTGGCAAGTCTCTTGGATC 19TCAAATGCCACTGAACCCTTTGCCAGATCAAGTTCAAGCTACAA CACGGGTTT amiRNA0192AGCTCCTTGTTAGGTTGGTGTTGATCTGGCAAGTCTCTTGGATC 20TCAAATGCCACTGAACCCTTTGCCAGATCAAGTCCAACCTACAA CACGGGTTT amiRNA0243AGCTCCTTGTTATCTTCGTGTTGATCTGGCAAGTCTCTTGGATC 21TCAAATGCCACTGAACCCTTTGCCAGATCAAGTCGAAGATACAA CACGGGTTT amiRNA0309AGCTCCTTGTTGACAGGATGTTGATCTGGCAAGTCTCTTGGATC 22TCAAATGCCACTGAACCCTTTGCCAGATCAAGTTCCTGTCACAA CACGGGTTT amiRNA0382AGCTCCTTGTTGAGGTCATGTTGATCTGGCAAGTCTCTTGGATC 23TCAAATGCCACTGAACCCTTTGCCAGATCAAGTTGACCTCACAA CACGGGTTT amiRNA0423AGCTCCTTGTTGGTATGGTGTTGATCTGGCAAGTCTCTTGGATC 24TCAAATGCCACTGAACCCTTTGCCAGATCAAGTCCATACCACAA CACGGGTTT amiRNA0458AGCTCCTTGTTGGGATCTTGTTGATCTGGCAAGTCTCTTGGATC 25TCAAATGCCACTGAACCCTTTGCCAGATCAACAAGATCCCACAA CACGGGTTT amiRNA0483AGCTCCTTGTTGTCAGATTGTTGATCTGGCAAGTCTCTTGGATC 26TCAAATGCCACTGAACCCTTTGCCAGATCAAGTATCTGACACAA CACGGGTTT amiRNA0514AGCTCCTTGTTGTCGTGATGTTGATCTGGCAAGTCTCTTGGATC 27TCAAATGCCACTGAACCCTTTGCCAGATCAAGTTCACGACACAA CACGGGTTT amiRNA0531AGCTCCTTGTTGTCTTCGTGTTGATCTGGCAAGTCTCTTGGATC 28TCAAATGCCACTGAACCCTTTGCCAGATCAAGTCGAAGACACAA CACGGGTTT amiRNA0569AGCTCCTTGTTGTGTGATTGTTGATCTGGCAAGTCTCTTGGATC 29TCAAATGCCACTGAACCCTTTGCCAGATCAACAATCACACACAA CACGGGTTT aMIR164-rps23-1AGCTCCTTGTTTCTCGGAAATTGCGCTTCCAAGTCTCTTGGATC 30TCAAATGCCACTGAACCCTTTGGAAGCGCAAAATCCGAGAACAA CACGGGTTT

Example 4 In vivo Transgenic Root-SCN Assays

Expression vectors containing target-specific artificial miRNAs weretransformed into soybean roots to test their ability to reduce SCN cystsas transgenes. Soybean cultivar Williams 82 was used as the germplasmfor the hairy root transformation. Seeds of soybean seeds weregerminated on 1% agar containing 0.5% sucrose in Petri dishes at 27° C.for 5 days. The cotyledons were then cut off the seedlings, and thewounded surface was inoculated with cultures of the Agrobacteriumrhizogenes carrying the binary vector. The cotyledons were placed on 1%agar for 6 days and then transferred onto selection media. In about twoweeks, independent transgenic hair root events induced from thecotyledons were harvested and transferred onto culture media, andcultured in the darkness at 27° C. Narayanan et al. indicated that SCNresistance phenotypes in the whole plant were preserved in transgenichairy roots, therefore transgenic hairy root system is useful forevaluating candidate SCN resistance genes. Narayanan et al. (1999) CropScience 39, 1680-1686.

Two weeks after transfer onto the culture plates, the transformed hairyroots were inoculated with surface-sterilized J2 stage soybean cystnematodes (SCN J2) and the roots were grown in darkness at 27° C., whichallowed cyst formation on the hairy root events. One month afternematode inoculation, the number of cysts was determined for both theroots expressing target-specific artificial miRNAs and the rootsexpressing the empty vector (as a negative control).

In this experiment, when the amiRNA0097, amiRNA0145, amiRNA0043,amiRNA0483, amiRNA0309, amiRNA0382, amiRNA0243 and amiRNA0514 vectorconstructs were overexpressed in the transgenic soybean hairy root, thecyst formations on these roots were significantly reduced compared tothe controls. The aMIR164-rps23-1 amiRNA was used as a positive controlbecause this miRNA had been shown to reduce SCN cyst formation. Althoughthe expression of the other amiRNAs such as amiRNA0046, amiRNA0569,amiRNA0458, and amiRNA0531 did not significantly reduce the number ofSCN cysts compared to the controls, the results do not indicate thatthese few amiRNAs are ineffective at targeting SCN RNAs. For example, animproved expression strategy that results in a higher level ofexpression of the siRNA might increase the efficacy of these siRNAs. Theassay results for amiRNAs reducing cyst formation in soybean hairy rootsare listed in the following tables.

TABLE 10 amiRNA Vector Sequences amiRNA ID Vector ID SEQ ID NOamiRNA0043 pKS49 31 amiRNA0046 pKS50 32 amiRNA0097 pKS100 33 amiRNA0145pKS101 34 amiRNA0192 pKS102 35 amiRNA0243 pKS105 36 amiRNA0309 pKS106 37amiRNA0382 pKS107 38 amiRNA0423 pKS62 39 amiRNA0458 pKS52 40 amiRNA0483pKS108 41 amiRNA0514 pKS109 42 amiRNA0531 pKS53 43 amiRNA0569 pKS51 44aMIR164-rps23-1 pKS104 45 Vector Control Empty 15312 46

TABLE 11 In vivo transgenic root-SCN assay Plasmid ID Gene of InterestAvg. Cysts n Standard error Empty Vector None 17.7 3 3.8 (SEQ ID NO: 46)(Negative Control) pKS104 aMIR164-rps23-1 10.4 5 1.1 (SEQ ID NO: 45)(Positive Control)

TABLE 12 In vivo transgenic root-SCN assay Plasmid ID Gene of InterestAvg. Cysts n Standard error Empty Vector None (Negative 34.1 7 3.2 (SEQID NO: 46) Control) pKS100 siRNA0097 11.1 11 1.2 (SEQ ID NO: 33) pKS101siRNA0145 16.3 16 1.8 (SEQ ID NO: 34) pKS49 siRNA0043 21.9 8 3.3 (SEQ IDNO: 31) pKS50 siRNA0046 32.2 5 4.4 (SEQ ID NO: 32)

TABLE 13 In vivo transgenic root-SCN assay Plasmid ID Gene of interestAvg. Cysts n Standard error Empty Vector None (Negative 16.5 10 2.1 (SEQID NO: 46) Control) pKS105 siRNA0243 11.6 8 2.1 (SEQ ID NO: 36)

TABLE 14 In vivo transgenic root-SCN assay Plasmid ID Gene of interestAvg. Cysts n Standard error Empty Vector None (Negative 33.0 4 4.8 (SEQID NO: 46) Control) pKS62 siRNA0423 29.0 8 4.7 (SEQ ID NO: 39) pKS51siRNA0569 32.2 4 5.2 (SEQ ID NO: 44)

TABLE 15 In vivo transgenic root-SCN assay Plasmid ID Gene of interestAvg. Cysts n Standard error Empty Vector None (Negative 25.0 5 5.4 (SEQID NO: 46) Control) pKS52 siRNA0458 26.0 6 4.0 (SEQ ID NO: 40) pKS53siRNA0531 21.7 3 3.5 (SEQ ID NO: 43)

TABLE 16 In vivo transgenic root-SCN assay Plasmid ID Gene of interestAvg. Cysts n Standard error Empty Vector None (Negative 35.6 11 3.0 (SEQID NO: 46) Control) pKS108 siRNA0483 17.5 13 2.0 (SEQ ID NO: 41) pKS109siRNA0514 19.5 22 1.2 (SEQ ID NO: 42)

TABLE 17 In vivo transgenic root-SCN assay Plasmid ID Gene of interestAvg. Cysts n Standard error Empty Vector None (Negative 78.1 10 6.7 (SEQID NO: 46) Control) pKS106 siRNA0309 51.7 16 7.4 (SEQ ID NO: 37)

TABLE 18 In vivo transgenic root-SCN assay Plasmid ID Gene of interestAvg. Cysts n Standard error Empty Vector None (Negative 154.9 7 11.0(SEQ ID NO: 46) Control) pKS107 siRNA0382 106.4 14 8.7 (SEQ ID NO: 38)

Example 4.1 Dual Activity of amiRNAs

Two of the amiRNAs tested above, amiRNA0097 comprising siRNA0097, andamiRNA0145 comprising siRNA0145 (See Table 12) caused an increase inroot growth and proliferation in the transgenic soybean cells in whichthey were expressed compared to soybean cells expressing the othersiRNAs or an empty-vector (negative control), suggesting that siRNA0097and siRNA01435 are modulating expression of one or more soybean genes inaddition to targeting a nematode gene. Although neither strand of any ofthe siRNAs tested above produced any full-length complementation to anysoybean genes when screened in silico against a soybean genome,surprisingly both strands of siRNA0097 and siRNA0145 have significantcomplementarity to two soybean orthologs of an Arabidopsis ethyleneresponse 1 gene (ETR1). Interestingly, ethylene receptor or responsegenes like ETR1-type genes have been implicated in root proliferationand an increased tolerance to some nematodes in certain plants. In onestudy, for example, chemically mutagenized soybean plants that wereethylene-insensitive (i.e. mutated ethylene response gene(s)) prodcuedabout 41% fewer females (cysyts) than wild-type non-mutagenized soybeanplants (Bent et al. 2006. Crop Science 46:893-901). To date, it does notappear that any studies have correlated RNA knock-out of an ethyleneresponse gene with nematode resistance.

Results of the siRNA strand complementation analysis are shown in Table19. Complementation was highest for both strands of amiRNA0097,particularly in the seed sequence (underlined sequence). The amiRNA0097plus strand has 7 out of 7 matches in the seed sequence to the sopybeanETR1 gene and the amiRNA0097* strand (star strand) has 6 out of 7matches in the seed sequence. amiRNA0145 plus strand has 5 out of 7matches and amiRNA0145* star strand has 6 out of 7 matches. Both ofthese treatments, amiRNA0097 and amiRNA0145 had enhanced reduction ofnematode infection (measured by cyst formation) compared to amiRNA0043that had only 4/7 mtaches to ETR1 and amiRNA0046 that had 5/7 matches toETR1 but which had a large gap between nucleotide 7 and 8.

TABLE 19 Complementation of siRNAs with Soybean   ETR1-type GenesComplementation alignment of siRNAs with  ETR1 mRNA

In recent years, it has been discovered that the miRNA* strand of someof the miRNA/miRNA* duplexes can also be loaded into the RISC andinterfere with the expression of its complementary mRNA target(Kulcheski et al, 2011. BMC Genomics 12:307). Therefore it is possiblethat the miRNA* can be loaded into the RNA-induced silencing complex(RISC) and used to silence the target mRNA. In addition to the plusstrand, both the amiRNA0097* and the amiRNA0145* star strands can formcomplementary binding with the gma-ETR1 mRNA (see Table 19 above),therefore it is possible that both strands of the amiRNA0097 andamiRNA0145 can down-regulate the expression of the gma-ETR1 gene.

Surprisingly, the results in Table 12 above show that soybean rootsexpressing the amiRNA0097 and amiRNA0145 had significantly enhancedresistance to cyst formation compared to the soybean roots expressingamiRNA0043, amiRNA0046 and the negative control (evident by thenon-overlap of their standard errors). The enhanced reduction in thenumber of cysts is likely due to siRNA0097 and siRNA0145 having both adirect effect on nematodes, i.e. siRNA0097 and siRNA0145 target anematode gene and suppress or silence that gene thus reducing the numberof cysts the nematode is capable of producing (See Example 2 above), andan indirect effect on nematodes, i.e. siRNA0097 and siRNA0145 alsosuppress expression of an endogenous plant gene (ETR1) by virtue oftheir having complementarity to ETR1 mRNA, which in turn confers someresistance to nematode infectivity (production of cysts). The enhancedeffect on cyst production then is likely due to the synergisticsuppression of a nematode gene and an endogenous plant gene. It isbelieved that this is the first report of such “direct” and “indirect”effect on nematode infectivity of a single siRNA molecule.

Example 5 Transformation of Plants with siRNAs

The artificial pre-miRNAs, gma-aMIR164-amir0097 andgma-aMIR164-amir0145, comprising the amiRNA0097/amiRNA0097* duplex andthe amiRNA0145/amiRNA0145* duplex, respectively (See Example 3), wereeach cloned into separate binary vectors and named 20111 and 20109,respectively. The 20111 and the 20109 vectors were transformed intosoybean.

Transformation of soybean to produce transgenic soybean plants wasaccomplished using immature seed targets of variety Williams 82 via A.tumefaciens-mediated transformation. Explant materials and media recipeswere essentially as described in Hwang et al. (PCT InternationalPublication No. WO 08/112,044) and Que et al. (PCT InternationalPublication No. WO 08/112,267), with some variations as noted below.Using this method, genetic elements within the left and right borderregions of the transformation plasmid are efficiently transferred andintegrated into the genome of the plant cell, while genetic elementsoutside these border regions are generally not transferred.

Maturing soybean pods were harvested from greenhouse-grown plants,sterilized with diluted bleach solution, and rinsed with sterile water.Immature seeds were then excised from seedpods and rinsed briefly withsterile water. Explants were prepared from sterilized immature seedsessentially as described in Hwang et al. (PCT International PublicationNo. WO 08/112,044) and infected with A. tumefaciens strain EHA101harboring either vector 20111 or 20109 and allowed to incubate for anadditional 30 to 240 minutes. Excess A. tumefaciens suspension wasremoved by aspiration and the explants were moved to plates containing anon-selective co-culture medium. The explants were co-cultured with theremaining A. tumefaciens at about 23° C. for about 4 days in the darkand then transferred to recovery and regeneration medium supplementedwith an antibiotics mixture consisting of ticarcillin (75 mg/L),cefotaxime (75 mg/L) and vancomycin (75 mg/L) where they are incubatedin the dark for seven days.

The explants were then transferred to regeneration medium containinghygromycin B (3 to 6 mg/L) and a mixture of antibiotics consisting ofticarcillin (75 mg/L), cefotaxime (75 mg/L) and vancomycin (75 mg/L) toinhibit and kill A. tumefaciens. Shoot elongation and regeneration wascarried out in elongation media containing 2-4 mg/L of hygromycin B. Thehygromycin phosphor-transferase (HPT) gene was used as a selectablemarker during the transformation process. Regenerated plantlets weretransplanted in soil essentially as described (PCT InternationalPublication No. WO 08/112,267) and tested for the presence of HPT andCMP promoter sequences using TaqMan PCR analyses (Ingham et al. (2001)Biotech 31, 132-140). This screen allows for the selection of transgenicevents that carry the T-DNA and are free of vector DNA. Plants positivefor HPT gene and CMP sequences and negative for the spectinomycin (spec)gene were transferred to the greenhouse for analysis of miRNA expressionand seed setting.

When the roots were about 2-3 inches, plants were then transplanted into1-gallon pots using Fafard #3 soil and 30 grams of incorporated OsmocotePlus 15-9-12 and maintained under standard greenhouse growing conditionsfor soybean plants.

The leaves of the transgenic soybean events of 20111 and 21019 weresampled to quantitatively determine the expression level of the ETR1gene using an art recognized quantitative real time polymerase chainreaction (qRT-PCR) (See for example, VanGuilder et al. (2008).Biotechniques 44 (5): 619-626; Udvardi et al. (2008). Plant Cell 20 (7):1736-1737). The relative amount of ETR1 gene expression in thetransgenic events and in wild-type control soybean plants was determinedby comparing to the ETR1 level to a different endogenous soybean gene.

Results of the qRT-PCR assays showed that ETR1 expression in thetransgenic soybean roots producing siRNA0097 and siRNA0145 wassignificantly lower compared to the wild-type control soybean roots. Therelative amount ETR1 expression level was about 34±5 (N=9, where N isthe number of plants) in wild-type soybean roots and about 23±4 (N=14,where N is the number of events) and about 12±1 (N=21, where N is numberof events) in the siRNA0145 and siRNA0097 transgenic soybean roots,respectively. Therefore, the siRNA0145 and siRNA0097 transgenic soybeanroots had a 33% and a 66% reduction in ETR1 gene expression,respectively, compared to the wild-type soybean roots.

These results correlate with the other results obtained for siRNA0097and siRNA0145 described above. siRNA0097 and siRNA0145 were designed totarget a nematode gene and upon contact of soybean cyst nematode witheither siRNA0097 or siRNA0145 the nematode's ability to produce cysts onsoybean roots was reduced (See Example 2). Soybean roots expressingsiRNA0097 or siRNA0145 had significantly reduced number of cysts wheninfected with SCN (Example 4) with siRNA0097 having a significantlylower number than siRNA0145. Interestingly, siRNA0097 had the highestcomplementarity in both strands to a soybean ETR1 gene and soybean rootsexpressing siRNA0097 had the lowest level of ETR1 gene expression. Bentet al. 2006 (supra) reported about a 41% reduction in cysts inethylene-insensitive soybean plants with a chemically mutated ETR1 gene.Here, soybean roots expressing an siRNA that directly targets an SCNgene and modulates the expression of an endogenous ETR1 gene had as highas a 68% reduction in the number of cysts, a level that is higher thanexpected of either the modulation of the nematode gene or the modulationof the ETR1 gene alone.

Although there have been reports of “off-type” effects of dsRNA designedto target a plant pest gene, to date, it appears that no studies havereported the suppression of an endogneous plant gene by an siRNAdesigned to target a gene of a nematode plant pest whereby thesuppression of the plant gene also confers resistance to the samenematode pest. Thus, it is surprising that an siRNA designed to target anematode gene suppresses the expression of both a nematode gene (directeffect on the nematode) and an endogenous plant gene that in turninterfers with nematode infectivity (indirect effect on nematode). It isfurther surprising that the modulation of an endogenous plant gene by asiRNA designed to target a nematode gene may be due to both the plus andstar strands of the siRNA.

Plants transformed with the vectors are inoculated with J2-stage soybeancyst nematodes (SCN J2). Briefly, 1-week old seedlings of the transgenicT1 generation soybean grown in germination pouches are inoculated withSCN J2 suspension at the level of 750 J2 per plant. One month afternematode inoculation, the number of cysts is determined for thetransgenic plants comprising amiRNA expression cassettes and for thenull segregants from the same TO parents.

Example 6 Corn Rootworm siRNA Library Design and Construction

A small interfering RNA library was prepared having a partiallyrandomized seed sequence to indiscriminately target mRNAs of an insectpest. The corn rootworm (CRW) was chosen as a target pest for testingthis siRNA library.

A 21-nucleotide small interfering RNA library was designed with arandomized seed sequence located at positions 2-8 from the 5′-end, andpositions 1, 9-21 were fixed. Since the small RNA was designed to targetinsect pest genes, the non-seed sequence was based on microRNAs from arelated coleopteran pest insect, Tribolium castaneum. Bioinformaticanalyses of the predicted and known T. castaneum miRNAs revealedconserved nucleotides at each position of the non-seed region of miRNAs(i.e., positions 1 and 11-19). These nucleotides were selected for thenon-seed sequence for the siRNA library. Uridine residues were chosenfor positions 20 and 21 in order to increase the stability of themolecule for in vitro screening. The model non-seed sequence generatedfrom the consensus T. castaneum miRNA is 5′-UNNNNNNNUAUCCGGAUUCUU-3′,(SEQ ID NO: 50) where N indicates a random nucleotide (i.e., either A,C, G, or U) in the seed sequence. A siRNA library of this exemplarysequence consists of 4⁷ (i.e., 4×4×4×4×4×4×4) different RNA molecules,or 16,384 possible sequences.

In order to reduce the complexity of an RNA library (i.e., the number ofsequences contained in the library), a subset of sequences were excludedfrom the library. In particular, the complexity of the siRNA library wasreduced by computationally excluding nucleotides that occurred at aparticular position in T. castaneum miRNA seed sequences at lowerfrequencies. In this example, the frequency threshold was chosen to be20%. Accordingly, any nucleotide that was determined to occur less than20% at a particular position in a T. castaneum seed sequence usingbioinformatic analyses was excluded at that particular position.Nucleotides that occurred with a frequency of 20% or greater in T.castaneum seed sequences were included in the library. Table 20 showsthe nucleotides that are frequently observed at each position.

TABLE 20 Nucleotides present in greater than 20% of T. castaneum miRNAseed sequences Position in miRNA seed sequence 2 3 4 5 6 7 8 Nucleotide1 A A A A A A A Nucleotide 2 C — C — — C C Nucleotide 3 G G G G G GNucleotide 4 U U U U U U U Number of Nucleotides 4 3 4 2 3 4 4 MotifSeed Sequence N D N W D N N

The reduced combination of nucleotides at the 7-positions within theseed sequence was equal to: 4×3×4×2×3×4×4, or 4608 possible sequences,which was a 3.6-fold reduction in complexity.

In addition, small RNA sequences were excluded from the in silicolibrary if the seed sequence contained homonulceotide quadruplets, suchas AAAA. Further, sequences having a GC-content in positions 1-9 (i.e.,position 1 and the seed sequence and position 9) greater than theGC-content of positions 11-19 were also excluded. After these twoadditional parameters were considered, the number of siRNA sequences inthe in silico library was reduced to 3899-sequences. The siRNA consensusmotif is 5′-UNDNWDNNUAUCCGGAUUCUU-3′ (SEQ ID NO: 51).

The 3899 siRNAs are synthesized as duplexes using standard automatedsynthesis. In order to enhance the stability, the 3′-residues may bestabilized against nucleolytic degradation, e.g., they consist of purinenucleotides. Alternatively, substitution of pyrimidine nucleotides bymodified analogues, e.g., substitution of uridine by 2′-deoxythymidineis tolerated and does not affect the efficiency of RNA interference.siRNAs can also be synthesized with a dTdT dinucleotide at the 3′-end asan overhang to increase stability and prevent nucleolytic degradation.

What is claimed is:
 1. A siRNA molecule comprising the nucleotidesequence of any one of SEQ ID NOs: 1-14 and wherein the siRNA moleculereduces the number of soybean cyst nematode cysts on soybean roots. 2.An artificial RNA molecule comprising the siRNA molecule of claim 1,wherein the artificial RNA molecule is encoded by the nucleotidesequence of any one of SEQ ID NOs: 16-29.
 3. A vector comprising thesiRNA molecule of claim 1, wherein the vector is encoded by thenucleotide sequence of any one of SEQ ID NOs: 31-44.